The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact, delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact, acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alpha (s1)-casein and beta -casein, showing very low activity towards kappa -casein. The BGPF1 proteinase completely hydrolysed only beta -casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.