A total of 1,102 clinical isolates of Gram-negative bacilli was obtained from four hospitals during 1967 and these cultures were tested for sensitivity to ampicillin. Approximately 80% of the strains of Escherichia coli and 90% of the strains of Proteus mirabilis, the two organisms most frequently isolated, were sensitive to ampicillin. Klebsiella-Enterobacter species and Pseudomonas aeruginosa were generally insensitive. Comparison of these results with data obtained in an earlier study with Gram-negative organisms isolated in 1961 showed that there had been no significant increase in the incidence of resistance of Gram-negative bacilli to ampicillin during the period 1961-67. The majority of ampicillin-resistant strains of E. coli isolated in 1967 transferred ampicillin resistance to a sensitive strain of E. coli K12. Only four ampicillin-resistant strains of E. coli isolated in 1961 were available for transferable resistance tests but all four strains transferred ampicillin resistance. Infective or transferable resistance was therefore a feature of ampicillin resistance of certain Gram-negative bacteria before ampicillin became generally available for clinical use.