Aim: Use of molecular techniques for the isolation of bacteria capable of phosphonoacetate mineralization as carbon, phosphorus and energy source. Methods and Results: RNA extracts obtained at three different stages of an enrichment selecting for phosphonoacetate degrading bacteria were reverse transcribed using 16S rRNA-specific primers, amplified and analysed by temperature gradient gel electrophoresis (TGGE). This information was used to devise a strategy for the isolation of members of the enrichment that were otherwise difficult to obtain in pure culture. We were able to pull out, in total, four out of the six main microbial cultures that were detected by TGGE. Two of the isolates belonging to Mycobacterium and Agromyces genera were for the first time shown to grow in the presence of phosphonoacetate as sole carbon, phosphorus and energy source releasing almost equimolar levels of inorganic phosphate into the culture medium, and they were shown to exhibit phosphonoacetate hydrolase activity in vitro. Conclusions: The ubiquity of pseudomonad in degradation processes is more likely a consequence of our ignorance of bacterial requirements and physiology, rather than their possession of unique metabolic properties. Significance and Impact of the Study: RT-TGGE analysis can be used to guide the successful isolation of micro-organisms difficult to obtain by culture-dependent methods alone.