We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F_2α and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca² ]_i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca² ]_i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF_2α and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF_2α increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF_2α and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF_2α steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF_2α action. The lower efficacy of PGF_2α in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF_2α to up-regulate ET-1.