SUMMARY: Some factors which affect the results of carbon source utilization (CSU) tests were investigated with 187 strains of Gram-negative bacteria, chiefly non-fermentative. There was little difference between the results at 37 and 30°C. Repetition of tests showed that 5% of the results were non-reproducible. The variability was most pronounced with acetate, maleate and sucrose amongst the substrates and Loefflerella mallei amongst the bacteria. Prior induction of the bacterial enzymes before testing did not reduce variability nor show any other advantage. Analysis of the results by numerical taxonomic methods revealed differences in the groups of bacteria derived from the results of CSU tests compared with those derived from conventional biochemical test results. CSU tests readily provide a large number of taxonomic features with the advantage of a simple standardized testing procedure.