We investigated several potential methods to select for carcinogen-induced changes in N-acetoxy-2-acetylaminofluorene-treated normal human diploid fibroblasts, in an effort to isolate cells exhibiting the transformed phenotype. Treated cultures exhibited an extended but not indefinite life span, as well as an increased cloning efficiency in reduced calcium concentrations at 40 to 50 population doublings posttreatment. Morphologically altered foci in monolayer culture, the capacity to proliferate under reduced serum or calcium concentrations, or the ability to grow on irradiated 3T3 monolayers did not uniquely identify or select for a carcinogen-induced phenotype. Treated cultures did routinely produce viable colonies when assayed under anchorage-independent (AI) conditions. This AI phenotype persisted for at least 2 months; when cells from such colonies were isolated and retested, a 2-fold enhancement in the frequency of AI growth was observed. AI-derived cells showed no stable morphological alteration in monolayer culture as regards either growth pattern or cytology. Four out of 10 strains of cells derived from AI colonies and grown to sufficient numbers in monolayer for tumorigenicity testing produced tumors in nude mice; only one of these was invasive and grew progressively to greater than 1 cm in diameter. Cells recovered from these tumors were diploid and of fibroblastic morphology. The AI phenotype appears to be an early marker for a carcinogen-induced change in human fibroblasts, but it is not systematically associated with the other phenotypic characteristics of transformation usually found concomitantly in rodent cell systems.