Gap junction-mediated intercellular communication (GJIC) is critical for maintaining integral cellular processes including differentiation and growth control. The disruption of GJIC has been correlated with aberrant function in many cell types, including hepatocytes in vivo; therefore it is imperative that cellular model systems support intercellular communication to simulate normal cellular functions. Functional GJIC has been shown in long-term primary rat hepatocyte cultures, which have been implemented widely to study various aspects of hepatocellular function; however, the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte system. In this report, we analyze GJIC in a dimethylsulfoxide (DMSO)-containing long-term primary mouse hepatocyte culture system. The cells retain morphologic and biochemical characteristics of differentiated hepatocytes through day 30 post plating, including liver-specific gene expression. We further show that connexin32 and connexin26 expression and gap junction plaque formation increase over time in culture concomitant with an increase in GJIC between adjoining primary mouse hepatocytes. In conclusion, the findings described in this study make it possible to maintain differentiated primary mouse hepatocytes that also show GJIC in long-term culture for 30 days. In addition, this system has the potential to be extended to study primary mouse hepatocytes isolated from genetically engineered mice.