Features of the transport system for hydroxyproline in a strain of Pseudomonas putida were studied. A mutant, lacking hydroxyproline-2 epimerase and unable to metabolize hydroxy-l-proline, was shown to transport and accumulate this compound after induction. Both entry and exit rates were examined, and kinetic constants for the reaction were determined. Increasing the induction time from 0.5 to 3 hr increased the entry rate three- to fourfold but had only a small and variable effect on the exit rate. Entry followed saturation kinetics. For hydroxy-l-proline, the K(m) and V(max) values were found to be 3 x 10(-5)m and 6 mumoles per g (dry weight) per min, respectively. The K(m) and V(max) for the epimer allohydroxy-d-proline were 10(-3)m and 0.1 mumole per g (dry weight) per min. Entry rates into "loaded" and "unloaded" cells were found to be the same. Exit was shown to be first order over the range of internal substrate concentrations measured. Exit rates were measured by several different methods and found to be independent of external substrate concentration. The first-order exit rate constant was computed to be 0.23 min(-1). Several metabolic inhibitors were examined for their effect on transport. The inhibitory action of N-ethyl maleimide was shown to be greatly reduced if cells were allowed to accumulate hydroxy-l-proline before exposure to the inhibitor. A number of other amino acids interfered with the transport of hydroxy-l-proline; the greatest effect was produced by l-alanine and l-proline.