The extracellular and cell-associated hydrolase profiles of a number of Pseudomonas fluorescens strains were examined with the Analytab API ZYM system. Esterase/lipase was the only strong extracellular enzyme activity detected (mean 3.33): weak esterase, lipase, and leucine aminopeptidase activities were found with some strains (mean activities of 1.08, 1.53, and 1.40, respectively). Very strong leucine aminopeptidase activity (4.5) was associated with the cells. Cell-associated trypsin, esterase/lipase, acid phosphatase, and phosphoamidase were also found. Neither extracellular nor cell-associated hydrolase profiles changed significantly when cells were grown in skim milk or mineral salts medium at either 5 or 20 degrees C. Similarly, added calcium did not seem required for synthesis of any of the enzymes. The extracellular enzyme profiles differed considerably from those of the cell-associated enzymes for all strains tested. An extracellular proteinase-deficient mutant of strain 32A (RM14) failed to produce significant quantities of extracellular esterase/lipase activity. Production of cell-associated enzymes was unaffected by the mutation. These results suggest that the Analytab API ZYM system may be useful in identifying psychrotrophs isolated from milk.