Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile-salt-dependent lipase (BSDL) was partly but specifically associated with endoplasmic reticulum membranes. In microsomes, temperature-induced phase separation using Triton X-114 elucidated the partition of BSDL between the aqueous phase and the detergent-rich phase containing hydrophilic and membrane proteins, respectively. The size of the membrane-associated BSDL (approx. 100 kDa) is compatible with that of the fully processed enzyme. Fucosylated O- and N-linked oligosaccharide structures were detected by means of specific lectins. The membrane-associated BSDL might therefore be released from membranes between the trans-Golgi compartment (where terminal fucose residues were added) and the zymogen granules where BSDL was mainly found in the soluble fraction. Even though BSDL associated with membranes was enzymically active, it appeared less efficient than the soluble form. The association of BSDL with membranes was pH-dependent and optimal association occurred between pH 5-6. The membrane-associated BSDL was released by KBr which suggests that the association of BSDL with microsomal membranes involves ionic interactions. Lipid-protein interactions are probably not involved in this association as BSDL did not associate with liver microsome membranes. We attempted to characterize the putative ligand and showed that BSDL and a 94-kDa protein, immunologically related to a glucose-regulated protein of 94 kDa (Grp94), were co-immunoprecipitated by specific antibodies directed against each individual species. It is suggested that the biogenesis of the human pancreatic BSDL involves an association with intracellular membranes and that its folding may be assisted by molecular chaperones.