Although carnitine palmitoyltransferase (CPT) has received considerable attention, particularly its regulation by malonyl CoA, most studies have monitored the forward reaction, ie, the formation of acylcarnitine. We examined the opposite or reverse reaction, in which palmitoyl CoA is generated, in osmotically-disrupted rat hepatic mitochondria. Specifically, the effects of pH, fasting, and untreated recent-onset diabetes were investigated. As with the forward (f) reaction, the CPT reverse (r) velocity v pH curve was somewhat parabolic with a pH maximum at approximately 7.2 (except the CPT that was from the diabetic rats). However, as the pH rose, the CPT reverse and forward curves diverged due to a precipitous decline in the forward reaction. This discordance in rates in the alkaline range was apparent in all three groups of CPT but was most prominent in the diabetic preparation (for example, as the pH increased from 7.3 to 8.8, the respective declines in the f and r velocites were 74% and 2%). In addition, under our assay conditions the CPTr from diabetic rats not only had a higher velocity (55.4 ± 1.4 nmol/min/mg protein) than that from the fed (32.1 ± 3.1) or fasted (43.1 ± 3.4) animals, but also the Vmax was found to be twofold greater, even though there was no difference in the Km for palmitoylcarnitine. In summary, diabetes affects the kinetics of the reverse reaction and, regardless of the animal's premortem condition, but more so in the diabetes, this reaction is less attenuated than the forward one as the pH rises. © 1988.