Growth and proteolytic activity of Lactococcus lactis strains were investigated in milks subjected to different heat treatments. Only 13·7% of the strains were of proteolytic phenotype. Proteinase-positive (Prt+) strains exhibited a proteolytic activity that increased when the level of heat treatment was reduced and grew to a higher level than proteinase-negative (Prt−) strains in low-heat-treated milk. When grown in autoclaved reconstituted skim milk, both Prt− and Prt+ strains had the same specific growth rate. Total DNA from four Prt+ and eleven Prt− strains was extracted and used for polymerase chain reaction amplification of fragments belonging to genes encoding for cell wall proteinase. Specific amplification products were displayed by all the Prt+ strains, and by the Prt− strain Lc. lactis subsp. lactis NWC 125. Reverse transcriptase-polymerase chain reaction was performed to detect the transcript of the proteinase genes. In addition to the Prt+ strains, the experiment also detected the specific transcript in Lc. lactis subsp. lactis NWC 125, suggesting that other structural or functional deficiencies occurred in this strain.