The authors describe their experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was use and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the Isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackleviruses. A seasonal effect on viral isolation rates from sludge was observed.