The acclimation or adaptation of microorganisms to organic chemicals is an important factor influencing both the rate and the extent of biodegradation. In this study two acclimation procedures were evaluated in terms of their effectiveness in enhancing biodegradation, their relative ease of use in the laboratory, and the implications for biodegradability testing. In the single‐flask procedure, microorganisms were acclimated for 2 to 7 d in a single acclimation flask at constant or increasing concentrations of the test chemical without transfer of microorganisms. In the second procedure, the enrichment procedure, microorganisms were acclimated in a series of flasks over a 21‐d period by making adaptive transfers to increasing concentrations of the test chemical. Acclimated microorganisms from each procedure were used as the source of inoculum for subsequent biodegradation tests in which carbon dioxide evolution was measured. Six chemicals were tested: quinoline, p‐nitrophenol, N‐methylaniline, N,N‐dimethylaniline, acrylonitrile, and 2,2,4‐trimethyl‐1,3‐pentanediol monoisobutyrate. Microorganisms acclimated in the single‐flask procedure were much more effective than those acclimated in the enrichment procedure in degrading the test chemicals. The single‐flask procedure is more convenient to use, and it permits monitoring of the time needed for acclimation. The results from these studies have implications for the methodology used in biodegradation test systems and suggest caution before adopting a multiple‐flask, enrichment acclimation procedure before the performance of standardized tests for aerobic biodegradability.