Lateral gene transfer by phages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency and range of phage-mediated gene transfer, it is important to understand the movement of DNA among microbes. Using an in situ DNA amplification technique (cycling primed in situ amplification-fluorescent in situ hybridization; CPRINS-FISH), we examined the propensity for phage-mediated gene transfer in freshwater environments at the single-cell level. Phage P1, T4 and isolated Escherichia coli phage EC10 were used as vectors. All E. coli phages mediated gene transfer from E. coli to both plaque-forming and non-plaque-forming Enterobacteriaceae strains at frequencies of 0.3-8 x 10(-3) per plaque-forming unit (PFU), whereas culture methods using selective agar media could not detect transductants in non-plaque-forming strains. The DNA transfer frequencies through phage EC10 ranged from undetectable to 9 x 10(-2) per PFU (undetectable to 2 x 10(-3) per total direct count) when natural bacterial communities were recipients. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viability in most cases. These results indicate that the exchange of DNA sequences among bacteria occurs frequently and in a wide range of bacteria, and may promote rapid evolution of the prokaryotic genome in freshwater environments.