Corpora lutea (CL) from synchronously ovulated, prepubertal rats remained viable in perifusing Eagle medium (Dulbecco modified) flowing at a rate of 1 ml/hr. As an index of viability, progesterone (P) and 20alpha-hydroxypreng-4-ene-3-one (20alpha-OH-P) were determined by radioimmunoa-say in effluent media. Corpora lutea removed on the 2nd day of diestrus (D-2) or the 2nd day of pseudopregnancy (PP-2) showed fatigue by a continuous decline in the concentrations of effluent progestins. P decreased from 2 to 0.2 ng/ml/CL during an interval of 5 hr, while 20alpha-OH-P decreased from 2.2 to about 0.7 ng/ml/CL. In contrast corpora lutea taken on PP-4 maintained higher progestin levels in the effluent media. P present initially at 2.2 ng/ml/CL decreased to 0.73 ng/ml/CL; 20alpha-OH-P, initial concentration 0.84 ng/ml/CL, decreased to 0.41 ng/ml/CL. The greater functionality of PP-4 CL indicated by the continued predominance of , suggested an in vivo imposition of a luteotropic stimulus occurring after PP-2. Addition of bovine LH (NIH-LH-B8, 20 mug/ml) to the perifusing medium stimulated steroidogenesis by PP-2 CL during which 20alpha-OH-P levels remained between 2.3 and 3.2 ng/ml/CL throughout perifusion, while P secretion decreased from 2.9 to 0.9 ng/ml/CL. Prolactin (NIH-P-B3, 20 mug/ml), did not significantly alter the original secretion pattern of PP-2 CL. During combined prolactin (20 mug/ml) and LH (2 to 10 mug/ml) perifusion, P secretion predominated, decreasing from 3.9 to 1.0 ng/ml/CL; 20alpha-OH-P decreased from 1.2 to 0.7 ng/ml/CL.