Connexins (Cx) are structural proteins that form gap junctions, which are vital to cell–cell communication and help to regulate cell division. The purpose of this study was to evaluate if there are diagnostically important differences in immunostaining for connexins 43 (Cx43) and 26 (Cx26) in melanoma compared with nevi. Formalin-fixed paraffin-embedded sections of 34 histologically well-characterized melanocytic lesions, 17 primary malignant melanomas (MM), and 17 nevi were stained with a polyclonal antibody to Cx43 and a polyclonal antibody to Cx26. Immunoreactivity in tumor cells was evaluated semiquantitatively based on extent (1%–100%) and intensity (0–3) of reactivity. A score of 0–300 was generated by the product of the extent and intensity readings in each case. Significantly higher Cx43 immunoreactivity was detected in MM (mean intensity score = 253.5; 95% confidence interval, 227.9–279.2; P = 0.002) compared with nevi (mean intensity score = 152.4; 95% confidence interval, 104.9–199.8). In contrast, Cx26 immunoreactivity was less than 5% or entirely absent in all melanocytic tumors (n = 34). The significantly higher Cx43 staining in MM when compared with nevi suggests an oncogenic role for this protein in melanocytic tumor progression. Consequently, the evaluation of immunohistochemical staining for Cx43 in conjunction with other ancillary stains and tumor histology may be helpful in distinguishing MM from nevi, although positive Cx26 reactivity suggests that a cutaneous neoplasm is of nonmelanocytic origin.