Incubation of rat jejunal slices in Krebs-Ringer bicarbonate buffer (KRB) required the presence of heat-inactivated horse serum (HHS) in order to show time-dependent release of sialyltransferase into the medium. Sialyltransferase activity could not be detected in the medium when KRB alone or KRB supplemented with either albumin or glycerol was used in the incubations. The viability of the jejunal slices for up to 4 h of incubation was determined by studying the incorporation of glucosamine and leucine into acid-insoluble proteins. Supplementation of KRB with HHS had no beneficial effect on the rate of incorporation of leucine and glucosamine into proteins. KRB medium obtained after different periods of incubation contained higher trypsin-like activity than KRB medium containing HHS. Various antiproteases present as supplements to KRB resulted in the release of sialyltransferase activity from the jejunal slices. Among these antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) was the most effective. Also, HHS added to KRB immediately following incubation resulted in partial restoration of sialyltransferase activity in the medium, suggesting the presence of anti-proteolytic factors in HHS. The addition of increasing concentrations of heparin to incubations containing HHS caused a decrease in the medium sialyltransferase activity. The heparin-binding fraction (HBF) from HHS, when added to incubations, was able to protect the sialyltransferase released into medium. However, HHS depleted of its heparin-binding fraction by heparin-agarose affinity chromatography was unable to protect the sialyltransferase. HBF was separated into high- and low-molecular-mass fractions (fractions A and B respectively) by gel-filtration chromatography. The capacity to protect the released sialyltransferase was contained in fraction B. Fraction A contained multiple bands on SDS/PAGE and did not protect the enzyme. Fraction B contained a major protein band on the gel which corresponded to the migration of a similar band in human alpha 1-PI. HBF as well as fraction B isolated from HHS showed anti-trypsin-like activity. The results presented indicate that HHS contains a heparin-binding protein(s) similar to human alpha 1-PI which plays a role in the protection of sialyltransferase released from jejunal slices.