An attempt was made to separate estrogen from prolactin dependency of 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors at 2.5 and 5 months after DMBA injection. Ovariectomy and drug and/or hormone treatments were used to produce an estrogen or prolactin deficiency for 2 weeks, followed by a 2-week period in which the deficiency was corrected. Tumors were classified as estrogen or prolactin dependent based upon regression in the absence of the hormone and resumption of growth upon hormone replacement. At 2.5 months and 5 months after DMBA injection, about 29 and 33% of the tumors, respectively, were classified as prolactin dependent, and 35 and 45%, respectively, were classified as estrogen dependent. However, the percentage of estrogen-dependent tumors was reduced to 2.2 and 9.7%, respectively, when prolactin levels were maintained after ovarierctomy. These results indicate that most DMBA-induced mammary tumors in Sprague-Dawley female rats are dependent on both estrogen and prolactin but that ovariectomy or estrogen administration do not accurately reflect estrogen dependency, since prolactin secretion also is altered by these procedures.