Carbon isotopic enrichment factors (epsilon(c)) measured during cometabolic biodegradation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by Pseudonocardia tetrahydrofuranoxydans strain K1 were -2.3 +/- 0.2 parts per thousand, -1.7 +/- 0.2 parts per thousand, and -1.7 0.3 parts per thousand, respectively. The measured carbon apparent kinetic isotope effect was 1.01 for all compounds, consistent with the expected kinetic isotope effects for both oxidation of the methoxy(or ethoxy) group and enzymatic S(N)1 biodegradation mechanisms. Significantly, delta C-13 measurements of the tert-butyl alcohol and tert-amyl alcohol products indicated that the tert-butyl and tert-amyl groups do not participate in the reaction and confirmed that ether biodegradation by strain K1 involves oxidation of the methoxy (or ethoxy) group. Measured hydrogen isotopic enrichment factors (CH) were -100 +/- 10 parts per thousand, -73 +/- 7 parts per thousand, and -72 +/- 2 parts per thousand for MTBE, ETBE, and TAME respectively. Previous results reported for aerobic biodegradation of MTBE by Methylibium petroleiphilum PM1 and Methylibium R8 showed smaller EH values (-35 parts per thousand and -42 parts per thousand, respectively. Plots of Delta H-2/Delta C-13 show different slopes for strain K1 compared with strains PM1 and 138, suggesting that different mechanisms are utilized by K1 and PM1/R8 during aerobic MTBE biodegradation.