Previously, this laboratory has demonstrated that azidothymidine used in combination with methionine enkephalin, an opioid pentapeptide, was more effective than AZT alone in inhibiting disease progression due to murine retrovirus infections. In order to study the mechanism(s) by which Met-ENK mediates-antiviral effects, when used in combination with AZT in Friend leukemia virus infected mice, an in vitro focus forming assay was used. AZT at 1 ng/ml inhibited FLV replication by 30-50% in the susceptible Mus dunni cell line. By contrast, the immunostimulatory neuropeptide, Met-ENK, displayed no direct inhibition of viral replication. This suggests that Met-ENK does not have any direct anti-retroviral activity. Subsequent testing of Met-ENK in the presence of AZT showed no ability of this peptide to promote inhibition of viral replication due to AZT. By contrast, in the presence of mouse spleen cells, as a source of lymphocytes, in vitro combination treatments using AZT and Met-ENK reduced FLV replication by 67%, compared to 47% using AZT alone. The inhibition due to Met-ENK was abrogated when spleen cells were pretreated with naloxone, an opioid antagonist. Therefore, we conclude that Met-ENK effects are mediated via opioid receptors on spleen cells and that the observed anti-FLV activity is dependent on the use of Met-ENK stimulated spleen cells in combination with AZT.