Glycerol uptake and glycerol kinase activity were studied in primary cultures of rat hepatocytes in the presence of either 1 nM insulin, 1 nM glucagon, or 100 nM dexamethasone, alone or in combination in the culture medium. Glycerol uptake exhibited saturation kinetic with K(m) values (microM) and Vmax (nmol/min x mg protein) ranging from 250-402, and 7.9-10.1, respectively. The corresponding K(m) and Vmax values for glycerol kinase activity were 36-46 and 8.7-12.7. Using the metabolic uncoupler 2,4-dinitrophenol, glycerol uptake and the cellular content of glycerol phosphorylated metabolites were reduced 33% and 43%, respectively, whereas no decrease in the cellular content of glycerol was seen. The glycerol analogues monoacetin, monobutyrin and dihydroxypropyl dichloroacetate were able in a concentration-dependent manner to inhibit glycerol uptake into hepatocytes with the two latter having IC50 values of approximately 1 mM. Moreover, it was demonstrated that the three glycerol analogues were substrates for glycerol kinase, which indicates a competitive mode of inhibition. The kinetic parameters for these substrates were calculated by using glycerol kinase from Candida Mycoderma. Monobutyrin was found to be 4 times lees efficient as substrate compared to the other substrates. Overall, these results indicate that independently of the culture conditions, glycerol uptake is the rate-limiting step in glycerol metabolism, and that the investigated glycerol analogues are metabolized via the same route as glycerol.