L-Carnitine uptake and exodus was studied in rat extensor digitorum longus muscle in vitro. A saturable transport process was observed, which had an apparent Km of 60 micron and V of 22 nmol/h per g tissue. Transport was inhibited by 2,4-dinitrophenol, sodium azide, anaerobiosis, ouabain, and sodium ion depletion. Analogs of L-carnitine containing a quarternary ammonium group were found to inhibit uptake (D-carnitine, Ki = 400 micron; gamma-butyrobetaine, Ki = 60 micron, choline chloride, Ki = 14 mM), while those not containing this functional group (gamma-aminobutyrate, D,L-beta-hydroxybutyrate) had no significant effect at concentrations 100 times the apparent Km of L-carnitine. Carnitine exodus from rat extensor digitorum longus muscle consisted of two phases. The rapid initial phase was attributed to leakage of L-carnitine from damaged muscle fibers, as it proceeded at nearly the same rat at 0 degrees and 37 degrees C, and then leveled off to a rate of near zero after 1 h of incubation in vitro. The quantitatively more important phase of exodus showed a latency of 1-2 h and then proceeded at a linear rate of 40-45 nmol/h per g tissue. The results of this study support the contention that L-carnitine is taken up by a carrier-mediated, active transport system in rat extensor digitorum longus muscle. Functionally, the transport system for uptake is distinct from the process by which carnitine is lost from this muscle.