The expression of 19 connexin (Cx) isoforms was observed in the mouse embryonic stem (ES) cell line, EB3. Their expression patterns could be classified into either pluripotent state-specific, differentiating stage-specific, or non-specific Cxs. We focused on Cx30.3 as typical of the first category. Cx30.3 was pluripotent state-specific and upregulated by leukemia inhibitory factor (LIF), a specific cytokine that maintains the pluripotent state of ES cell, via a Jak signaling pathway. Cx30.3 protein was localized to both the cell membrane and cytosol. The dynamic movement of Cx30.3 in the cell membrane was suggested by the imaging analysis by means of overexpressed Cx30.3-EGFP fusion protein. The cytosolic portion was postulated to be a ready-to-use Cx pool. The Cx30.3 expression level in ES cell colonies dramatically decreased immediately after their separation into single cells. It was suggested that mRNA for Cx30.3 and Cx30.3 protein might be decomposed more rapidly than mRNA for Cx43 and Cx43 protein, respectively. These indicate possible involvement of Cx30.3 in the rapid formation and/or decomposition of gap junctions; implying a functional relay between Cx30.3 and other systems such as adhesion proteins.