A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca² ]_i) and a protein kinase C-epsilon (PKCε)-specific inhibitor were used to investigate the developmental role of PKCε in the prostaglandin F_2α(PGF_2α)-induced rise in [Ca² ]_i and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF_2α increased [Ca² ]_i in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 ± 0.6, n = 116 vs. 21.3 ± 2.3, n = 110). Similarly, the fold increase in the PGF_2α-induced rise in [Ca² ]_i in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 ± 0.2, n = 198 vs. 2.7 ± 0.1, n = 95). A PKCε inhibitor reduced the PGF_2α-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 ± 0.3 (n = 217) and 1.3 ± 0.1 (n = 205), respectively. PGF_2α inhibited LH-stimulated progesterone (P₄) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCε-specific inhibitors reversed the ability of PGF_2α to decrease LH-stimulated P₄ accumulation, and the PKCε inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCε, an isozyme expressed in corpora lutea with acquired PGF_2α luteolytic capacity, has a regulatory role in the PGF_2α-induced Ca² signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF_2α to inhibit LH-stimulated P₄ synthesis at this developmental stage.