In situ hybridization was used to investigate the presence of mRNAs of three members of the serine protease inhibitor (serpin) superfamily, angiotensinogen (AG), alpha 1 protease inhibitor (alpha 1PI), and antithrombin III (ATIII) in normal human liver. The probes were full length 35S radiolabeled complementary DNAs of human AG, alpha 1PI, and ATIII. The three mRNAs were found to be uniformly distributed in all hepatocytes, with no evidence of any special distribution, but the signal was more intense for alpha 1PI than for AG and ATIII. Kupffer cells, biliary epithelial cells, and vascular cells were all negative. The same tissue was studied by peroxidase-antiperoxidase immunohistochemistry using specific antibodies against AG, alpha 1PI, and ATIII. No significant amounts of any of the proteins, alpha 1PI, AG, or ATIII were detected in frozen or fixed sections of normal liver. This study indicates that these proteins are not stored in the normal human hepatocyte, but that their genes are actively expressed and that in situ hybridization is the only technique presently available to detect their presence.