TPDMT-4 is a pregnancy-dependent mouse mammary tumor line characterized by progressive growth in pregnant, but dormancy in virgin, hosts and by significant estrogen (ER) and progesterone receptor (PR) levels. Three sublines, T4-OR26, T4-0I96, and T4-0I320, were established from sporadic outgrowths in virgins of TPDMT-4 pieces passaged long in breeding and estradiol (E2)- plus progesterone-treated mice and of enzymatically dissociated TPDMT-4 cells, respectively. T4-OR26 tumors produced ovarian-responsive growth and maintained the parent levels of ER and E2-dependent PR. T4-OI320 and T4-OI96 tumors were ovarian independent or autonomous. The former had ER, but not E2-dependent PR, and the latter had neither. To clarify the mechanism of acquisition of autonomy by hormone-dependent neoplastic cells, comparative ER studies were conducted between TPDMT-4 and T4-OI320 tumors. Although cytosolic, microsomal, and nuclear translocated ER levels were generally lower in T4-OI320 tumors, the dissociation constants, determined by the dextran-charcoal technique, steroid specificity, sedimentation data in sucrose gradients, and activation studies using DNA binding, molecular transformation from 4S to 5S, and [3H]E2 dissociation from [3H] E2-ER complexes as markers, revealed no autonomy-specific changes in cytosolic ER. Small but significant differences in the affinity of [3H]E2 to nuclear ER and the [3H]E2 dissociation rate from nuclear bound [3H]E2-ER complexes were found between both tumors. Hormone-dependent tumors may progress toward autonomy with different receptor statuses in different environments, and lack of E2-dependent PR synthesis in ER-positive tumors may be due to defects at postreceptor or nuclear levels involving subtle changes in interaction between activated ER and nuclei.