We have generated novel conditionally replicative adenoviruses (CRAds) targeted to melanoma cells. In these adenoviruses, the E4 region (AdΔ24TyrE4) or both E1 and E4 regions (Ad2x Tyr) were controlled by a synthetic tyrosinase enhancer/promoter (Tyr2E/P) specific for melanocytes. The properties of these CRAds were compared with wild-type adenovirus (Adwt) and our previous CRAd with a targeted E1A CRII mutation (AdTyrΔ24) in submerged cultures of melanoma cells and nonmelanoma control cells. We showed that AdΔ24TyrE4 had a cell type selectivity similar to AdTyrΔ24 but had a distinct block in viral reproduction in nonmelanoma cells and that Ad2x Tyr had an augmented selectivity for melanoma cells. These viruses were additionally tested in organotypic cultures of melanoma cell lines, primary human keratinocytes (PHKs), or mixed cell populations. Unexpectedly, the CRAds exhibited somewhat different cell type selectivity profiles in these cultures relative to those observed in submerged cultures, demonstrating the importance of multiple assay systems. Specifically, AdTyrΔ24 and Ad2x Tyr were selective for melanoma cells, whereas AdΔ24TyrE4 exhibited no selectivity, similar to Adwt. AdTyrΔ24 and Ad2x Tyr were strongly attenuated in their ability to lyse PHKs in organotypic cultures. Furthermore, Ad2x Tyr had a superior melanoma selectivity in organotypic cultures of cocultivated melanoma cells and PHKs. The enhanced selectivity for melanoma cells exhibited by Ad2x Tyr provides a window of opportunity for therapeutic application. These studies also demonstrate that organotypic cultures derived from mixtures of tumor and normal cells represent a promising new model for analysis of CRAd specificity and toxicity.