It is assumed that bacteria generally degrade 2-methylphenanthrene (2MPhe) in preference to 1-methylphenanthrene (1MPhe), and that environmental biodegradation of methylated PAHs therefore can be described qualitatively by changes in relative concentrations of these isomers. Our objective was to investigate whether microbial phenanthrene degraders (Sphingomonas and Mycobacterium) show such isomer-specific PAH degradation. Eleven out of twenty-nine phenanthrene degraders could grow on methylphenanthrene. The mycobacteria grew only on 2MPhe, the sphingomonads grew mostly on 1MPhe, and one sphingomonad could utilize both substrates. Seven strains were tested in a two-phase system where 1MPhe and 2MPhe were supplied in heptamethylnonane. For these strains, a consistent description of biodegradation based on the 2MPhe/1MPhe diagnostic ratio would not be possible because three Mycobacterium and one Sphingomonas degraded 2MPhe faster than 1MPhe, another Sphingomonas degraded 1MPhe and 2MPhe at almost equal rates, and two Sphingomonas degraded 1MPhe than 2MPhe. Thus, environmental biodegradation of phenanthrenes may theoretically proceed with only minor changes in 2MPhe/1MPhe ratios if individual members of the degrader community have different isomer preferences. However, two soil microcosms polluted with bunker oil confirmed the general decline in 2MPhe/1MPhe ratio during oil biodegradation.