The protein profiles of Pseudomonas putida F1 during growth on toluene, phenol, and their mixture were examined by two-dimensional polyacrylamide gel electrophoresis. Although this bacterium uses the same catabolic pathway for both substrates, P. putida F1 produced specific sets of proteins in response to toluene and phenol as single or mixed substrates. Proteins associated with growth on these substrates could be classified into three categories: ten Group T proteins were associated with the degradation of toluene, seventeen Group P proteins were associated with the degradation of phenol, and one Group M protein was observed to be associated only with toluene-phenol mixture degradation. During growth on the mixture, the protein profile of the cells shifted from Group T proteins to Group P proteins. This correlated well with the substrate consumption pattern, in which toluene was consumed first and growth on phenol did not begin until the medium was nearly depleted of toluene. Individual Group T and Group P protein intracellular concentrations had different transients as the cells grew on the mixture; seven protein levels increased, four decreased, and sixteen reached a maximum and then declined. The Group M protein reached a concentration maximum near the time when growth on phenol began. Variations in the maintenance of these proteins were also noted. These results demonstrate that cells growing on a mixture of substrates undergo significant physiological changes. Further investigation of these changes is expected to shed light on the unusual biodegradation kinetics previously observed with this mixed-substrate system.