Adenoviruses are among the most persistent and ubiquitous viruses in water and wastewater, but some of them are difficult to detect due to non-cytopathogenicity and slow growth in cell cultures. A TaqMan real-time RT-PCR method in conjunction with cell culture infectivity was developed to rapidly detect mRNA produced by infectious adenoviruses in water samples. Only infectious adenoviruses were detected by this method, based on their ability to produce mRNA during replication in cell culture. The mRNA of Ad41 was detected as soon as 3 days after infection at levels as low as 5 infectious units (IU) per cell culture. In order to confirm that our methods detected only infectious viruses, 1-ml volumes of 10(4)IU of Ad41 were exposed to different free chlorine doses. No mRNA was detected in cells inoculated with Ad41 treated with the highest free chlorine dose of 100 mg min/l. However, mRNA of adenovirus was detected in cells inoculated with virus that was untreated or exposed to a lower free chlorine dose of 1 mg min/l. These results suggest that mRNA detection by real-time RT-PCR is a sensitive and specific method to detect low levels of infectious adenoviruses in water and other environmental media within 1-3 days.