The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin El was measured as a function of temperature between 13 and 33°C, using a thermostatted flow dialysis system. Complete inhibition of proline transport at 33 and 130C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin El at an effective multiplicity of about 4. At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures. Further studies were carried out at 150C. Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15°C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20%. The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin El that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport. The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel. It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin El.A sensitive cell population treated with lethal doses of colicin will show a higher survival level if trypsin is added to the cell suspension a short time after the colicin has adsorbed to the cell surface. Because the biochemical targets of some colicins reside in the inner membrane and trypsin acts only on protein exposed at the external surface of the outer membrane, the trypsin experiment can provide information on the topographical arrangement of colicin in the cell envelope during the inhibitory stage. The first description of trypsin reversal of the inhibitory effect of a colicin was that of Nomura and Nakamura (24), using colicin K. The demonstration that trypsin, added at times long after the addition of colicin K, could result in substantial restoration of the colicin-inhibited rate of synthesis of ,B-galactosidase and nucleic acids (24) led to the concept that colicin bound at the external surface of the outer membrane could exert an effect on the inside of the cell (20, 23).Several aspects of the original trypsin rescue experiments have been examined in subsequent work, including the length of time after colicin addition over which trypsin rescue can occur, the influence of cellular energy state on this time interval (12, 23, 27, 29, 31, 32), and the identity t Present address: Department of Microbiology, The University of Leeds, Leeds L52 9JT, England. of the irreversible colicin-induced physiological damage that determines the end of the period during which colicin-treated cells can be rescued by trypsin (9,16,22,26,29,30). One question which remains from all these studies is whether trypsin can reverse co...