Connexin 36 (Cx36) is the predominant connexin isoform expressed in the mammalian neurons of the central nervous system (CNS). PC-12 cells, a neuronal-like cell line, are widely used for neuron functional studies. Many connexins have been shown to interact with zonula occludens-1 protein (ZO-1), a tight junction associated with protein. The present study is intended to investigate whether Cx36 is expressed in PC-12 cells and is associated with ZO-1. Cx36 transcripts were amplified and verified by RT-PCR. 2.9 kb Cx36 mRNA was detected in PC-12 cells through Northern blot hybridization. Western blotting showed a 36-kDa protein band in the homogenates of PC-12 cells. Immunofluorescence labeling revealed that Cx36 was present in cell-cell contacts of PC-12 cells and colocalized with ZO-1. The association of Cx36 and ZO-1 in PC-12 cells was also demonstrated by coimmunoprecipitation. In conclusion, PC-12 cells express Cx36 mRNA and Cx36 proteins that are associated with ZO-1. These results enhanced our understanding of the function of Cx36 in PC-12 cells.