We have examined melanocytic cells derived directly from fresh biopsy tissue for the presence of p53 mutations. Using selective media that permits growth of melanocytes and inhibits growth of fibroblasts and keratinocytes, we established short-term, primary cultures of melanocytes from skin biopsies of common acquired nevi, dysplastic nevi, and from metastatic melanoma. Using PCR-single-stranded conformational polymorphism analysis, we have detected p53 mutations in 2 of 11 benign compound nevi and 2 of 5 dysplastic nevi. All nevi positive for p53 mutations were derived from patients who previously had cutaneous moles and three of the four had a family and/or personal history of melanoma.