We have developed four new mammary adenocarcinoma cell lines from the C3(1)/SV40 Large T-antigen (Tag) transgenic mouse model: M28N2 and M27H4 (weakly tumorigenic), M6 (carcinoma), and M6C (metastatic). The C3(1) promoter directs Tag expression to the mammary epithelium and 100% of female C3(1)/Tag transgenic mice develop mammary adenocarcinoma in a predictable and progressive manner. The cell lines we developed from this model are demonstrated to be of epithelial origin and display growth rates, both in vitro and following subcutaneous inoculation into nude mice, that are consistent with their representative stage of tumor progression. The more tumorigenic cell lines, M6 and M6C, both express the sodium/iodide symporter, a mammary carcinoma cell marker with potential therapeutic and diagnostic applications. All of the cell lines express estrogen receptor (ER) alpha and ER beta mRNA, and Western blot analysis demonstrates that the ER alpha protein is down-regulated in the M6 and M6C cell lines. M28N2 cells also express progesterone receptor (PgR), which is very unusual in a mouse mammary carcinoma cell line. In addition, all of the cell lines display growth inhibition when plated in media supplemented with charcoal-stripped fetal calf serum (CS FBS). When CS FBS is supplemented with beta estradiol or the progestin MPA, no significant difference in growth rates is observed relative to growth in CS FBS. The development and characterization of a progressive series of new mammary carcinoma cell lines will aid in the study of mammary carcinoma progression both in vitro and in vivo.