We have examined whether assimilation of CO2 can be used as a measure of metabolic activity in planktonic and sessile heterotrophic bacteria. CO2 assimilation by environmental samples and pure cultures of heterotrophic bacteria was studied using 14CO2 and 13CO2 as tracers. Heterotrophic growth on complex organic substrates resulted in assimilation of CO2 into cell biomass by activated sludge, drinking water biofilm, and pure cultures of Escherichia coli ATCC 25922, Es. coli ATCC 13706, Rhodococcus ruber, Burkholderia sp., Bacillus circulans, Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonas aeruginosa. Analysis of 13C-labelled phospholipid fatty acids (PLFAs) confirmed that heterotrophic bacteria may assimilate 13CO2 into cell macromolecules such as membrane lipids. All major PLFAs extracted from activated sludge and drinking water biofilm samples were enriched in 13C after incubation with CO2. Between 1.4% and 6.5% of the biomass produced by cultures of P. putida and a drinking water biofilm during growth in complex media was apparently derived from assimilation of CO2. Resting cells assimilated less CO2 compared to actively growing cells, and CO2 assimilation activity correlated with the amount of biomass produced during heterotrophic growth. The 14CO2 assimilation assay was evaluated as a tool to examine inhibitory effects of biocides on planktonic and sessile heterotrophs (biofilms). On the basis of 14CO2 assimilation activity, the minimum inhibitory concentration (MIC) of benzalkonium chloride was estimated to 21.1 and 127.2 mg l(-1) for planktonic and biofilm samples, respectively. The results indicate that assimilation of isotopically labelled CO2 can be used as a relatively simple measure of metabolic activity in heterotrophic bacteria. CO2 assimilation assays may be used to study the effects of antimicrobial agents on growth and survival of planktonic and sessile heterotrophic organisms.