Recent technical developments have enabled the application of compound-specific isotope analysis (CSIA) of low (parts per billion) concentrations of 1,4-dioxane that are often found in groundwater at 1,4-dioxane-contaminated sites. However, to quantify 1,4-dioxane biodegradation, isotopic enrichment factors are needed to interpret the CSIA data obtained from field samples. In this study, the carbon and hydrogen isotopic enrichment factors (eC and eH, respectively) for 1,4-dioxane biodegradation have been determined for axenic propane- or isobutane-grown cultures of Rhodococcus rhodochrous ATCC 21198 and for tetrahydrofuran-grown cultures of Pseudonocardia tetrahydrofuranoxidans K1. The enrichment factors for propane-grown (eC = −2.7 ± 0.3‰, and eH = −21 ± 2‰) and isobutane-grown (eC = −2.5 ± 0.3‰, and eH = −28 ± 6‰) cells of strain 21198 were similar and substantially smaller than those determined for tetrahydrofuran-grown cells of strain K1 (eC = −4.7 ± 0.9‰, and eH = −147 ± 22‰). The presence of 1-butyn...