Hydrolysis of retinyl esters in the lumen of the small intestine is required before absorption. Previously, rat brush border membranes (BBMs) were found to contain a pancreatic-derived esterase preferring retinyl esters with short fatty acyl chains and an intrinsic esterase preferring esters with long fatty acyl chains. Here, similar activities were found for preparations of human BBMs. Long-chain ester hydrolysis was stimulated best by deoxycholate, a dihydroxy bile salt, whereas short-chain ester hydrolysis was stimulated best by taurocholate, a trihydroxy bile salt. A 10-fold difference in KM values for retinyl palmitate (0.53 mumol/L) and caproate (5.5 mumol/L) also indicated distinguishable long-chain and short-chain activities. Differences between retinyl butyrate and retinyl caproate hydrolysis suggested the possible presence of two short-chain esterase activities associated with both rat and human BBMs, in addition to the long-chain activity. Similarities between human BBM retinyl ester hydrolytic activities and those observed for rat BBMs suggest that the rat is a good model for humans in this step of vitamin A metabolism.