Cell lines originating from cattle, sheep, goat, deer, bison, swine, rabbit, hamster, cat, dog, monkey, human, and mosquito were obtained from the American Type Culture Collection and tested for contamination with bovine viral diarrhea virus (BVDV). Immunocytochemical procedures and polymerase chain reaction (PCR) amplification were used to detect viral antigen or viral RNA in 13 of 41 cell lines. The results of these procedures correlated exactly. Cell lines derived from cattle, sheep, goat, deer, bison, rabbit, and domestic cat were found contaminated with BVDV. Attempts were made to experimentally infect 14 swine, rabbit, hamster, cat, dog, monkey, and human cell lines that had been found free of virus. All swine cell lines, and most rabbit and cat cell lines, became infected with BVDV. Hamster, human, dog, and certain rabbit and cat cells were refractory to BVDV infection. Experimental infection of monkey cells produced variable results.