Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is the intracellular enzyme responsible for the esterification of cholesterol with long-chain fatty acyl-CoA derivatives and has been implicated in atherosclerosis and gallstone disease. The effects of exogenous cholesterol and dithiothreitol (DTT) on the ACAT activity of human liver microsomes have been determined. Pre-incubation of microsomes with exogenous cholesterol gave a marked stimulation of activity. Experiments with [3H]cholesterol and [14C]oleoyl-CoA indicated the time course of equilibration of exogenous with endogenous cholesterol as ACAT substrates, and showed that ACAT activity could be accurately measured using [3H]cholesterol/Tween 80, providing that the concentration of endogenous microsomal cholesterol was also determined. Pre-incubation of liver microsomes for 90 min in the presence of 2 mmol/l DTT and exogenous cholesterol/Tween 80 resulted in a 60% reduction in ACAT activity, compared with the corresponding activity when DTT was omitted. If microsomes were pre-incubated with DTT prior to the pre-incubation with exogenous cholesterol/Tween 80, an 85-90% reduction in ACAT activity occurred. In contrast, pre-incubation of microsomes with DTT in the absence of exogenous cholesterol/Tween 80 (only endogenous cholesterol present) resulted, initially in a stimulation of ACAT activity; on further pre-incubation, activity returned to control levels. These results indicate that the supply of cholesterol to the enzyme active site is an important factor in ACAT assays in vitro and that DTT has a major effect on this process, suggesting that these factors may be important in controlling ACAT activity in vivo.