Cell lines are used for the production of pharmaceutical proteins such as antibodies, and sophisticated steps are employed in order to clear viral contamination from the final product. The recent occurrence of a new xenotropic retrovirus derived from xenografting has drawn attention to the dissemination of xenotropic mouse (and related) retroviruses. We therefore investigated human and animal cell lines, which are reported to be free of exogenous retroviruses, by using multi-primer ultrasensitive reverse transcription together with the polymerase chain reaction (RT-PCR) to detect murine C-type retroviruses. Several mouse myeloma cell lines were found to release retroviruses. These cell lines all have P3K cells derived from BALB/c tumor MOPC21 as their common ancestor. Successful infection of mink cells, but not of mouse and human cells, indicated a xenotropic host range. Full length sequence analysis of the isolated and cloned viruses revealed distinct homology with known xenotropic mouse retroviruses, except for a domain in the p12/CA boundary of the Gag gene. Despite a low virus release rate in tissue culture, an unexpected outburst of viruses was observed in fermentation runs with Sp2/0 myeloma cells. These findings are of relevance for the safety of protein products derived from myeloma cells.