Cutaneous nodular lesions caused by parapoxvirus infections are commonly observed in stranded pinnipeds following their arrival at rehabilitation facilities. An indirect enzyme-linked immunosorbent assay (ELISA) was developed and validated to determine exposure to parapoxviruses in California sea lions Zalophus californianus in captivity and in the wild. The diagnostic performance of this assay was evaluated using receiver-operating characteristic analysis. At a selected cut-off value, the calculated sensitivity was 100% (95% CI = 86 to 100%) and the specificity was 100% (95% CI = 87 to 100%). Analysis of sera collected from 26 affected sea lions during various stages of the disease revealed anti-parapoxvirus antibodies in all affected sea lions prior to the development of cutaneous pox lesions. This indicated that previous exposure to a parapoxvirus does not confer protection against clinical disease. In at least 7 cases, exposure to the virus occurred during hospitalization. Analysis of paired sera from 74 unaffected sea lions indicated subclinical infections in at least 3 animals. Finally, the prevalence of anti-parapoxviral antibodies in 761 free-ranging California sea lions captured and tested was 91% (95% CI = 89 to 93%). This indicated that infection with a parapoxvirus is a common occurrence in the wild and that the release of captive sea lions infected with parapoxvirus into the wild should not increase the risk of a parapoxvirus outbreak in free-ranging sea lions.