A murine malignant melanoma cell line, PAZG, resistant to 300 µg 8-azaguanine (AZG)/ml was isolated from mass cultures of B16 cells exposed to increasing analogue doses for more than 2 years in vitro. PAZG cells proliferated in medium containing high AZG concentrations and, because of a complete deficiency in hypoxanthineguanine phosphoribosyltransferase (HGPRT), did not incorporate exogenous guanine or hypoxanthine. Three observations supported the hypothesis that this enzyme deficiency was a stable, genetic cell characteristic: 1) PAZG cells either transferred to, and subcultured for 76 days in, medium lacking AZG or passaged through host animals still retained resistance to the analogue; 2) no revertants were found in 107 resistant cells; and 3) PAZG cells, fused with murine cells resistant to 5-bromodeoxyuridine and lacking thymidine kinase, formed hybrids which contained active HGPRT as well as thymidine kinase and proliferated in medium containing hypoxanthine, aminopterin, thymidine, and glycine. Studies of features such as morphology, karyology, and tumorigenicity failed to disclose any detectable differences between AZG-sensitive and AZG-resistant cell lines.