In an effort to develop a genetic therapy for the treatment of breast cancer, we constructed adenoviral vectors containing either the beta-galactosidase (beta-gal) reporter gene or the herpes simplex thymidine kinase (HSVtk) suicide gene driven by breast tissue-specific promoters. We utilized upstream regulatory sequences from either the human alpha-lactalbumin (hALA) gene, or the ovine beta-lactoglobulin (oBLG) gene in these vector constructs to target expression of heterologous genes transcriptionally to breast cancer cells both in vitro and in vivo. Data derived from breast tissue-specific reporter vectors in vitro demonstrate that expression from the hALA and oBLG promoters are indeed specific for breast cells (T47D, MCF-7, ZR75-1) when compared with non-breast cells (U2OS, HeLa). Moreover, these vectors displayed tumor cell specificity when compared with the normal MCF-10A breast cell line. These vectors also displayed breast tissue specificity when injected systemically (i.v.) into lactating Balb/c mice, which suggests that these promoters maintain their tissue-specific expression pattern within the context of the adenoviral genome in vivo. Tumors, derived from T47D human breast cancer cells, were established in nude mice and injected with either the tissue-specific reporter or suicide vectors. Results from tumors injected (i.t.) with reporter adenoviruses demonstrate that these promoters are active in T47D cells when grown as established tumors and we observed a marked regression of tumors injected with suicide vectors and treated systemically with gancyclovir (150 mg/kg/day) when compared with control animals. Moreover, mouse survival was prolonged after 35 days in mice undergoing therapy with the suicide vectors in conjunction with gancyclovir when compared with the control animals. These data suggest that the transcriptionally targeted hALA or oBLG driven expression of the HSVtk gene may be a feasible therapy for the treatment of human breast cancer.