The mechanism of tetracycline resistance mediated by R931 (a Pseudomonas aeruginosa R factor not yet successfully transferred to Escherichia coli recipients) was examined. In strain 931 (R931) (minimal inhibitory concentration [MIC] 200 mug/ml) significant tetracycline uptake did not occur until 100 mug of tetracycline per ml was included in uptake studies. The introduction of R931 into strain 280 resulted in a significant decline in (3)H-tetracycline uptake. In both strains 931 (R931) and 280 (R931), a further reduction in tetracycline uptake resulted from pre-incubation with 1 mug of tetracycline per ml. Tetracycline resistance in R(-)P. aeruginosa strains 1731, 1885, and 494, considered to be of chromosomal origin, was associated with a lack of tetracycline uptake until the MIC of the strain was obtained. No evidence of tetracycline inactivation or ribosomal resistance was detected in R(-) or R(+) strains. The MIC for R(-) strains was generally about 25 mug/ml and that for R(+) strains was 75 to 200 mug/ml.