Plasma levels of prolactin (PRL) were determined before and during suckling in intact and ovariectomized (OVX) lactating rats treated 5 days earlier with a single sc injection of 50 micrograms polyestradiol phosphate (PEP) or saline. Radioimmunoassay (RIA) and the Nb2 lymphoma cell bioassay (BA) were used to measure plasma PRL. Pituitary PRL concentrations were also determined by the two assays. In addition, lactational performance was estimated by measuring litter weight gain before and after estrogen treatment. Lactation was inhibited by PEP in intact rats; however, PEP did not alter plasma or pituitary levels of PRL in this group. Lactation was not inhibited by estrogen in OVX rats, but plasma PRL levels were significantly reduced at 10, 30, and 45 min of suckling. In addition, there was no evidence of an estrogen-induced afternoon prolactin surge in either PEP-treated group. The lactational inhibition in the intact, PEP-treated rats was probably due to combined effects of estrogen and progesterone. Plasma progesterone concentration was 64 +/- 8 ng/ml in the intact PEP-treated group compared to 3 +/- 1 ng/ml in the OVX, PEP-treated rats. The exact mechanism for the supression or delay in suckling-induced plasma PRL in the OVX, PEP-treated females remains unresolved but it was not due to an increase in suckling-induced corticosterone levels. The qualitative changes in plasma PRL during suckling in the various groups detected by RIA were also detected by the BA. However, there were slight quantitative differences between the two assays. When plasma PRL was low (nonsuckled states) the BA/RIA ratio was less than that observed when the plasma PRL was high (during suckling), i.e., more bioactive PRL was apparently released by suckling. However, these differences between RIA and BA may have been due in part to the fact that very low or very high levels of prolactin approached the minimal and maximal assay limits of the RIA but not the BA indicating that care be exercised when making such assay comparisons.