We are designing and synthesizing rigid guests to probe the topography of the carnitine acyltransferases, regulatory enzymes in lipid metabolism. Our designs are based on structural studies of substrates and possible molecular mechanisms of enzymatic activity. Recent X-ray, 1H NMR, and force-field computational studies on carnitine and acetylcarnitine, coupled with the known stereospecificity for activity in carnitine acyltransferases, have led us to propose a molecular mechanism for acyl transfer in these enzymes. The ‘folded’ conformation of an acylcarnitine is most populated and should be preferred for binding to these enzymes, because, in this conformation, the acyloxy is the most sterically accessible. There are four key recognition sites on the enzymes: I, carboxylate; II, trimethylammonium; III, coenzyme A; IV, acyl. Sites, I, II and III serve as the three loci required to create a chiral environment on the enzymes for carnitine. An addition-elimination reaction involving the formation of a tetrahedral intermediate is suggested as the mechanism for O-to-S acyl transfer. This proposed tetrahedral intermediate is chiral and the enzymes should prefer the R configuration at this center. Based on this proposal, conformationally rigid tetrahedral-intermediate analogues have been designed, synthesized and assayed. Morpholinium and 2-hydroxymorpholinium derivatives inhibit carnitine acetyltransferase and palmitoyltransferase. Because of rigidity at their two chiral centers, these inhibitors serve as probes of molecular topography of recognition sites, I, II, and IV.