This study developed and evaluated the RT-PCR detection of mRNA in cell culture to assay infectious Adenoviruses (Ads) using Adenovirus types 2 (Ad2) and 41 (Ad41) as models. Only infectious Ads are detected because they are the only ones able to produce messenger RNA (mRNA) during replication in cell culture. Three primer sets for RT- PCR amplification of mRNA were evaluated for their sensitivity and specificity for the detection of Ads types 2/5 and 40/41. The first primer set targets a conserved region of late mRNA transcript (for the hexon region encoding a virion structural protein), and it detects a wide range of human Ads, including types 2 and 5 (respiratory and enteric viruses) and type 40 and 41 (enteric viruses). Two other primer sets target a region of an early mRNA transcript (in the E1A region of the viral genome) that specifically detects either Ads 2 and 5 or Ads 40 and 41. Following primer optimization, A549 and Graham 293 cell cultures were infected with Ad2 and Ad41, respectively. The mRNA of infected cells was recovered from cell lysates using Oligo-dT at different time periods (6 hours - 10 days) after infection. The recovered mRNA was treated with RNase-free-DNase to remove residual contaminating DNA and then Ad mRNA was detected by RT-PCR. The study detected mRNA (both E1A and hexon) of Ad2 as early as 6 hours after infection at higher inocula levels and after longer incubation times, at levels as low as 1-2 IU per cell culture. It also detected mRNA (both E1A and hexon) of Ad41 as soon as 24 hours after infection and at levels as low as 5 IU per cell culture. In order to confirm that these methods detect only infectious viruses, the study exposed 104 IU of Ad2 and 102 IU of Ad41 to different CT doses (0, 1, 30, 100 mg*min/L) of free chlorine. The study detected no mRNA in cells inoculated with Ads treated with any CT dose of free chlorine (1, 30, 100 mg*min/L), however, as expected, mRNA of Ads was detected in cells infected with untreated virus. The study also exposed 5 105 IU of Ad2 and 103 IU of Ad 41 to different doses (0, 50, 100, 250 mJ/cm2) of collimated, monochromatic (254 nm) UV radiation. The study detected no mRNA in cell cultures inoculated with Ads exposed to the highest UV dose (250 mJ/cm2) whereas mRNA was detected in cell cultures inoculated with untreated virus (zero UV dose) and at the lower (50 and 100 mJ/cm2) UV doses. In experiments on both free chlorine and UV disinfection, detection of mRNA of Ad2 exactly coincided with the presence of virus infectivity detected by cytopathogenic effects (CPE) in A549 cell cultures. These results indicate that mRNA detection by RT- PCR in inoculated cell cultures is a very sensitive and specific method to detect infectious Ads. Includes 27 references, tables, figures.