The study of growth of endometrial cells is of importance in reproductive biology. Several factors and hormones are thought to play important roles in the control of growth. Prostaglandin F2 alpha (PGF2 alpha) causes an increase in both tritiated thymidine ([3H]Tdr) incorporation into DNA and in the cell number of primary cultures of rabbit endometrial cells cultured in a serum-free, chemically defined media. Prostaglandins F1 alpha, E1, E2, A2, and B2 and arachidonic acid (all tested at 10(-7) M) do not affect [3H]Tdr incorporation as compared to control cultures. The increase in [3H]Tdr incorporation into DNA in response to PGF2 alpha stimulation is concentration-dependent (optimal approximately 3 X 10(-7) M) and is seen starting approximately 9 hr poststimulation. Both prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), but not PGs F1 alpha, I2, A2, B2, their parent molecules, or related molecules, antagonize and can completely block the PGF2 alpha-induced increase in [3H]Tdr incorporation into DNA. This antagonism is seen both when the cells are pretreated with PGE1 prior to the PGF2 alpha stimulation and when the cells are exposed to both PGE1 and PGF2 alpha simultaneously. Exogenously added 8-Br-cAMP mimics the PGE1 antagonism of PGF2 alpha. The PGF2 alpha-induced increase in [3H]Tdr incorporation is not synergistic, antagonistic, or additive with the [3H]Tdr incorporation increase in response to either estradiol-17 beta or epidermal growth factor. The specific effect of PGF2 alpha on primary culture endometrial cell growth and its antagonism by PGE1, PGE2, and 8-Br-cAMP are new findings.