The luteal phase in dogs is governed by many, poorly-understood regulatory mechanisms. Functioning of the CL is unaffected by hysterectomy. Recently, the role of prostaglandins in regulating canine CL function was addressed suggesting a luteotropic effect of PGE2 during the early luteal phase. However, compelling functional evidence was lacking. The potential of PGE2 to stimulate steroidogenesis was tested in canine primary luteal cells isolated from developing CL of non-pregnant dogs. In addition, the luteal expression of prostaglandin transporter (PGT) and steroidogenic acute regulatory protein (STAR) was demonstrated and characterized in CL from non-pregnant bitches during the course of dioestrus as well as from pregnant animals during the pre-implantation, post-implantation and mid-gestation periods of pregnancy and during luteolysis; the luteal expression of PGE2-receptors (EP2 and EP4) has been investigated at the protein level throughout pregnancy. Our findings show that PGE2 is an activator of STAR expression in canine luteal cells from early luteal phase, significantly upregulating STAR promoter activity and protein expression resulting in increased steroidogenesis. The 3βHSD- and P450scc-expression remained unaffected by PGE2 treatment. The expression of PGT was confirmed in CL both during pregnancy and dioestrus and generally localized to the luteal cells. After initial upregulation during the earlier stages of the CL-phase, its expression declined towards the luteal regression. Together with the demonstration of EP2 and EP4 throughout pregnancy, and the decline in EP2 at prepartum, our findings further support our hypothesis that intra-luteal PGE2 may play an important role in regulating progesterone secretion in the canine CL.