Signal transducer and activator of transcription (STAT3) plays a pivotal role in tumor progression. Atypical nevi are nonobligate risk markers of melanoma, affording investigators a target for evaluation of progression biomarkers in vivo. pSTAT1tyr701 (pSTAT1) and pSTAT3tyr705 (pSTAT3) oppose one another in biological function. Therefore, an analysis of phosphorylated STAT1 (pSTAT1) and pSTAT3 signaling was performed simultaneously using double-immunohistochemistry in biopsies of 168 atypical nevi from 42 patients receiving high- or low-dose IFNalpha (HDI and LDI). With maturation of melanocytes from junctional into dermal components of nevi, pSTAT1 expression increased, whereas pSTAT3 expression decreased. The percentage of pSTAT3-positive melanocytes was positively associated with the atypical degree of nevi (P<0.0001). In the junctional component of nevomelanocytic lesions, HDI and LDI downregulated the percentage of pSTAT3-positive melanocytes (P=0.008 and P=0.0003, respectively) while upregulating the percentage of pSTAT1-positive melanocytes (P=0.016 and P=0.0059, respectively) and augmented the pSTAT1/pSTAT3 ratio (P=0.008 and P=0.0040, respectively). It is suggested that the relative balance of pSTAT1/pSTAT3 may be associated with melanocyte differentiation in vivo. pSTAT3 is a potential biomarker of melanocytic transformation and progression and is modulated by IFNalpha dose-dependently. STAT3 is a potential target for chemoprevention of melanoma.