Objective: To test the hypothesis that DHEAS production from DHEA occurs in hepatic cells and that this production is augmented by the presence of sex steroids or insulin. Design: In vitro prospective experiment. Setting: Academic medical center. Intervention(s): Hepatoma G2 cells cultured in media supplemented with  DHEA (10-5 mol/L) only,  DHEA (10-5 mol/L) + T (10-6 mol/L),  DHEA (10-5 mol/L) + E2 (10-6 mol/L),  DHEA (10-5 mol/L) + dihydrotestosterone (10-6 mol/L),  DHEA (10-5 mol/L) + insulin (10 ng/mL), or  DHEA (10-5 mol/L) + insulin (100 ng/mL). Main Outcome Measure(s): Levels of DHEAS in the media were measured at 0, 2, 4, 6, 8, 12, 24, 48, and 72 hours after adding treatments at time-point 0. Result(s): Dehydroepiandrosterone sulfate was first detected in the hepatoma G2 cell culture media at 12 hours of incubation. The cumulative production rate of DHEAS increased linearly until 72 hours of incubation. When compared with the effect of treatment with DHEA only, treatment with DHEA plus T, dihydrotestosterone, or E2 delayed the cumulative DHEAS production; alternatively, the addition of insulin did not alter DHEAS production. Conclusion(s): These data suggest that although hepatic cells have the ability of converting DHEA to DHEAS, neither sex steroids nor insulin results in the increased hepatic production of DHEAS. © 2009 American Society for Reproductive Medicine.