We have synthesized (2S,6R:2R,6S)-6-carboxymethyl-2-hydroxy-2-pentadecyl-4,4-dimethylmorpholinium bromide (hemipalmitoylcarnitinium, HPC), which is a conformationally restricted analog inhibitor of carnitine palmitoyltransferase (CPT; EC 220.127.116.11). rac-HPC inhibits catalytic activity in purified rat liver CPT. In the forward reaction, HPC competes with both (R)-carnitine (Ki(app) = 5.1 ± 0.7 μM) and palmitoyl-CoA (Ki(app = 21.5 ± 4.9 μM). In the reverse reaction, inhibition by HPC is competitive with palmitoyl-(R)-carnitine (Ki(app = 1.6 ± 0.6 μM), but inhibition is uncompetitive with CoA. The forward reaction is also competitively inhibited by its product, palmitoyl-(R)-carnitine, Ki(app)'s 14.2 ± 2.1 μm relative to (R)-carnitine and 8.7 ± 2.6 μm relative to palmitoyl-CoA. rac-HPC is the most potent synthetic reversible inhibitor of purified CPT. HPC fails to inhibit carnitine acetyltransferase (CAT; EC 18.104.22.168). Palmitoylcholine also inhibits CPT in the forward reaction, competing with (R)-carnitine (Ki(app = 18.6 ± 4.5 μM) and with palmitoyl CoA (Ki(app = 10.4 ± 2.5 μM). Choline is not an effective CPT inhibitor. We have shown [R. D. Gandour et al. (1986) Biochem. Biophys. Res. Commun. 138, 735-741] that hemiacetylcarnitinium inhibits CAT but not CPT. The combined data demonstrate further differences between the carnitine recognition sites in CPT and CAT.