A putative fatty acid specificity of bile salt-dependent lipases (BSDLs) has been re-investigated. The strategy was to use two evolutionally distant, homologous BSDLs (from human and cod), and to investigate their hydrolysis of different fatty acid esters at different assay conditions affecting the physicochemical phase of the substrate. Depending on assay conditions, large variations were seen in the hydrolysis rate for esters of different fatty acids. The two enzymes displayed similar fatty acid specificity patterns, with small, but significant differences that were maintained at various assay conditions. Compared to the human enzyme, the cod enzyme showed a preference for hydrolysis of long-chain polyunsaturated fatty acyl esters (up to 22 carbons in length). On the other hand, the human enzyme hydrolysed esters of shorter chain saturated fatty acids at significantly higher rates compared to the cod enzyme. Changing physicochemical factors affecting the substrate phase induced large changes in fatty acid specificity that affected both enzymes in similar manners. It is concluded that though the aliphatic chains of the fatty acids may not be recognized by the enzymes, these chains indirectly affect the conformation or interfacial availability of the carboxyl ester bond in the substrate, and the enzymes show minor specificities for variations in these structures.