Abstract: Utilization of proline in Escherichia coli K-12 was analyzed at different osmolarities. Under the proline-base cultivation, sufficient growth of strain K-12 accompanied with high amount of proline uptake was observed in the presence of less than 0.5 M NaCl, where growth time lag was recognized. Distinct decrease in growth yield was observed in the presence of 1 M NaCl despite the sufficient accumulation of proline. Activities on respiration supported the result of growth. Analyses of L-[5-3H]proline uptake in the cell cytoplasm suggested that proline in cells was incorporated into hot TCA insoluble fraction in the absence of NaCl, but it was efficiently utilized as compatible solute in the presence of high concentrations of NaCl. These data suggested that proline itself functions as compatible solute rather than the participation of metabolization in cells under high osmolarity.