Assay of narrow fractions from three asphalts designated 1A, 3A, and 6A indicates the chemical nature of some of the asphaltic constituents utilised by Mycobacterium ranae and Pseudomonas 196Aa. These organisms were tested with pure hydrocarbons for growth response. The results indicated that Ps. 196A utilised n‐alkanes containing 6—22 carbon atoms, some alkenes and fatty acids but none of the aromatic hydrocarbons tested. M. ranae utilised substituted benzene compounds, but not benzene, alkenes or most alkanes. Using asphalt fractions obtained by thermal diffusion, infra‐red data were correlated with microbial results. The two approaches support each other in partial elucidation of the structures of the asphalt fractions.