Beginning in Lebanon Station at a channelized intersection with US 19/US 98 and County Road 336 in Levy County, SR 121 travels through Goethe State Forest where it turns east and intersects CR 337. The road makes a sharp left curve to the northeast where it travels through desolate farmland; the only resemblance to a major intersection is CR 326. Within the city of Williston, SR 121 has a concurrency with CR 316, until the intersection with US 41. It then joins US 41 as CR 316 continues eastward, which then joins the east end of US 27 Alternate, and then the US 27/US 41 concurrency before breaking away to the northeast again, taking the DeSoto Trail away from US 41. North of CR 335, SR 121 hugs the west side of the Levy–Marion county line, but never crosses it, choosing to enter Alachua County instead. Immediately after the interchange with Interstate 75 at exit 382, SR 121 makes a sharp left onto Northwest 34th Street as SR 331 becomes the designation for Williston Road. As it crosses SR 24 it borders the University of Florida to the east until crossing SR 26A and then SR 26. North of US 441, the DeSoto Trail breaks away. After the intersection with CR 236 in Santa Fe, SR 121 is carried along bridges across the former Seaboard Coast Line railroad line to Crystal River 3 Nuclear Power Plant and Santa Fe River in Worthington Springs, where it intersects SR 18. Briefly traveling parallel to SR 100 in Lake Butler, it intersects SR 100 as it continues northeast. The road also intersects SR 16 in Raiford, and I-10 and US 90 in Macclenny close to SR 228 before crossing the Georgia state line on its way to South Carolina.
From a point on SR 20 and SR 25 approximately northwest of Paradise in a northwesterly direction to the intersection with SR 235 in LaCrosse and along SR 235 for approximately, then continuing northwesterly via Santa Fe to a junction with SR 18 at Worthington Springs and along SR 18 to a point approximately north of Worthington Springs, then northeasterly to a junction with SR 100 in Lake Butler and East on SR 100 to a point near the eastern city limits of Lake Butler then northeasterly via Raiford to a junction with SR 10 in Macclenny and along SR 10 to 5th Street then in a northerly direction along 5th Street to the St. Mary's River at the Georgia state line.
Prior to the renumbering, this had been:
SR 99 from northwest of Paradise to Worthington Springs
At some point, SR 23 was renumbered as part of SR 121, probably to match Georgia State Route 121. Before that, however, SR 23 had been extended around the west side of Gainesville to end at US 441/SR 25 at Rocky Point, and the southernmost part did not become part of SR 121. It was later given to the county, and has a possibility of becoming County Road 23. The State Road 23 designation has since been reused for a partial beltway around Jacksonville.
Major intersections
County Road 121A
County Road 121A is the sole suffixed alternate of SR 121 that exists within the State of Florida. It is a former segment of SR 121 that exists entirely in LaCrosse. The route is unmarked, and runs along the east side of SR 121 south of downtown to halfway between the SR 121/235 overlap across from the LaCrosse Post Office.
