Connexin 43 (Cx43) is the most abundant gap-junction protein in ovarian follicle cell interactions and has been well characterized for several mammalian species. However, the involvement of Cx43 in canine follicular development has not been extensively investigated. The objective of this study was to 1) characterize the spatial patterns of Cx43 localization within the canine ovarian follicle at various physiological states and during the estrous cycle (prepubertal, anestrus, proestrus, estrus and diestrus), and 2) to characterize the canine Cx43 mRNA transcript. The spatial expression pattern of Cx43 protein was evaluated by immunofluorescence microscopy in canine ovaries. Cx43 was not detected in primordial follicles, but was detected in primary and secondary follicles at each physiological state at all granulosa cell borders. In secondary follicles, definitive, punctate staining patterns were localized along mural granulosa cells and also in the surrounding granulosa-cumulus cell borders. Notably, more intense staining was observed in the corona radiata cells immediately surrounding the oocyte, as well as in trans-zonal projections and at the perivitelline membrane. Patterns of localization were most similar between proestrus and diestrus, and between prepubertal and anestrus in secondary follicles. Estrus-stage follicles showed a decrease in localization at the corona-oocyte cell borders as compared to proestrus and diestrus. In large, healthy antral follicles from proestrus and estrus stages, Cx43 was present in the stroma, theca, and granulosa layers. However, antral follicles from estrus-stage ovaries showed more intense staining in the mural granulosa and theca layers, and less intense in the stroma as compared to those of proestrus stage. The most intense pattern of punctate staining was observed in the corpora lutea of diestrus-stage ovaries. Additionally, gene-specific primers were designed from highly conserved regions of Cx43 mRNA among bovine, human and mouse. RNA was isolated from canine uterus and used as a template for RT-PCR. The PCR products were then sequenced and verified in GenBank to assess homology. The sequenced coding region for canine Cx43 mRNA shares 93% sequence homology with bovine vascular smooth muscle. Canine specific primers for this sequence have been designed, and expression analysis studies in canine ovarian follicles are currently underway. These results indicate that the pattern of localization of Cx43 is similar to that reported for the cow and the pig, except that in canine ovarian preantral follicles, Cx43 is also localized to the peri vitelline membrane. Additionally, these results suggest that the localization of Cx43 is dependent on the physiological state of the ovary, and is likely necessary for folliculogenesis and subsequent oocyte development in canines.