Parvalbumin (PV) is a high-affinity Ca 2+-binding protein which is expressed in a limited number of vertebrates tissues and restricted to a few distinct cell types. It has been shown by biochemical methods to be present in the adult rat ovary, but cellular localizations or developmental appearance have not been described until now. This study describes the presence of PV and its transcripts, analyzed by immunohistochemistry and in situ hybridization, respectively, during the postnatal development of the rat ovary: 13 developmental stages between day 1 and day 83 were examined. In ovaries 1–16 days old, neither PV mRNA nor PV was observed in any cell of the ovary. By contrast, starting from day 18 postpartum, both PV mRNA and PV were detected in low amounts, simultaneously with the onset of differentiation of secondary interstitial gland cells in the ovarian interfollicular stroma. PV and its transcripts were primarily detected in conspicuous patches of interstitial gland tissue and in the differentiated thecal cells around the large follicles, and PV appeared to be fully expressed 33 days after birth. The parallel time courses of PV mRNA and PV accumulation during developmental differentiation, and the appearance of a steroid-producing cellular phenotype as well as the strict cellular colocalization of these two features, strongly suggest involvement of PV in the steroid metabolism of these cells, as earlier proposed for the Leydig cells of the testis. According to this hypothesis, we also show that suppression of gonadotrophic hormone production by hypophysectomy of adult rats totally suppresses PV production in parallel with the disappearance of the morphological features typical of steroid-producing cells in the remaining interstitial tissue of the ovary.