The activities of the DNA polymerases alpha, beta, and gamma were determined in rat giant trophoblast cells during mid-gestation. Trophoblasts at this period of time have ceased to divide but continue to carry out DNA endoreduplication resulting in polyploidy. DNA polymerase-alpha activity in extracts was found to drop sharply from a high level at Day 11 to 1/10 of that level at Day 12 and to continue at a constant level thereafter. A similar pattern of activity was observed for polymerase-gamma, however, in this case the drop represented 50% of the activity at 11 days. Polymerase-beta showed no significant change in activity during this period of development. Endoreduplication (polyploidy) continued during this period as measured by a linear increase in chromosomal DNA content. The sharp drop in alpha-polymerase activity from Day 11 to Day 12 appears to result from the loss of a protein(s) which specifically stimulate(s) alpha-polymerase.