Previous in vivo studies in the ovine fetus have demonstrated net serine production by the fetal liver, a pattern not seen in the adult sheep. The goal of this study was to determine the major metabolic pathways responsible for fetal hepatic serine production by using stable isotope methodology in primary culture of late gestation ovine fetal hepatocytes. Specifically selected tracers of glycine were added to individual cultures, with production of labeled serine determined after 24 h of incubation. When [1-13C1]glycine or [2-13C1]glycine was used as the initial tracer, serine enrichment at 24 h indicated that approximately 30% of serine production comes from glycine. Quantitative comparison of serine enrichment from these two tracers suggests that serine synthesis from glycine occurs via the combined action of the glycine cleavage enzyme system (GCE) and serine hydroxymethyltransferase (SHMT). Using [1,2-13C2(15)N1]glycine as the tracer, there was no significant increase in M + 2 glycine in the medium over 24 h, suggesting no reversible transamination of glycine, and therefore no significant movement of glycine through the glyoxalate pathway. These data demonstrate that in primary culture of fetal ovine hepatocytes, approximately 30% of serine biosynthesis is derived from glycine, primarily via the combined action of GCE and SHMT.