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In view of the widespread use of in vitro incubations of corpus luteum slices for a variety of purposes, experiments were conducted to establish optimum incubation conditions and to determine some of the sources of error variation observed in this system. A 2-hr incubation period was sufficient to detect an LH (luteinizing hormone) induced stimulation of progesterone synthesis; longer incubation times yielded higher levels of synthesis but did not increase the precision of the assay. The pH optimum for the incubation system was between 7.15 and 7.55. Addition of 1 μmole NADPH2 resulted in a consistent stimulation of progesterone synthesis, even in those groups already maximally stimulated with LH, suggesting that 2 different mechanisms are operative for the 2 stimulating agents. NADPH2 did not change the regression coefficient or the precision of the assay. Addition of nicotinamide (30 HIM) resulted in higher rates of progesterone synthesis, presumably by maintaining the integrity of the pyridine nucleoti...