The subunit structure of C8 is unusual in that it consists of a disulfide-linked alpha-gamma-dimer and a non-covalently associated beta-chain. To gain insight into the biosynthesis and assembly of C8, we examined its production by short term cultures of rat hepatocytes. Synthesis was detected by functional assays as well as immunologically with antisera against C8, alpha-gamma or beta. Control experiments showed that all three antisera were capable of precipitating C8 from rat serum. When antiserum against C8 was used to analyze hepatocyte cell lysates, precursor as well as mature forms of alpha-gamma and beta were detected. Precursor-product relationships were confirmed by pulse-chase experiments, which also revealed a significantly faster rate of synthesis for alpha-gamma. Importantly, no single chain form of alpha-gamma was detected in the intracellular pool. Evidence that C8 is assembled before secretion was provided by analyzing cell lysates with antiserum specific for alpha-gamma or beta. Precipitation of both subunits with either antiserum confirmed they were associated. Parallel analyses of the extracellular pool revealed that alpha-gamma and beta remain associated in the media but that excess alpha-gamma is secreted as a consequence of a faster synthesis rate. These results provide evidence for differential expression of alpha-gamma and beta and are consistent with the existence of different loci for these subunits. Furthermore, they indicate that association of alpha-gamma with beta is a presecretory event in normal C8 synthesis.