We examined the influence of connexin (Cx) expression on the development of apoptosis in HeLa parental cells (coupling deficient cell line) and HeLa cells expressing wild-type Cx43 and Cxs fused with enhanced green fluorescent protein (EGFP). EGFP was attached to the C-terminus of Cx32 and Cx43, Cx32-EGFP and Cx43-EGFP, respectively, and to the N-terminus of Cx32, EGFP-Cx32. All fusion proteins assembled into junctional plaques (JPs) at areas of cell-cell contact, but only the C-terminal fusion proteins formed functional gap junction (GJ) channels as well as hemichannels. In each cell line, apoptosis was induced by treatment with various agents including anisomycin, camptothecin, cis-platinum, colchicine, cycloheximide, etoposide, staurosporin and taxol. Using fluorescence microscopy, time-lapse imaging and dual whole-cell voltage clamp techniques, we correlated the changes in functional properties of GJ channels and Cx distribution with the progression of apoptosis based on cells' labeling with acridine orange and ethidium bromide (EB). The early phase of apoptosis (a viable apoptotic (VA) state) was characterized by shrinkage of the cells and by increased internalization of JPs accompanied by decreased cell-cell coupling. The apoptotic reagents had no direct effect on electrical cell-cell coupling. Transformation from a VA to a nonviable apoptotic (NVA) state was faster in HeLa cells expressing Cx43 or Cx43-EGFP than in HeLa parental cells. The potent GJ uncoupler, octanol, slowed the transition of HelaCx43-EGFP cells into a NVA state. In the absence of apoptotic reagents, the rate of EB uptake was higher in HeLaCx43-EGFP than in HeLa parental cells consistent with the presence of open Cx43-EGFP hemichannels. However, in both cell lines the rate of EB uptake decreased proportionally during the development of apoptosis suggesting that membrane permeability ascribed to Cx hemichannels is reduced. Cells expressing Cx32-EGFP and EGFP-Cx32 demonstrate the same apoptotic patterns as HeLaCx43-EGFP and HeLa parental cells, respectively. Intracellular levels of ATP in HeLaCx43-EGFP cells were substantially lower than in HeLa parental cells, and ATP added to the medium abolished the accelerated transition from a VA to a NVA state in HeLaCx43-EGFP cells. In summary, Cx32 or Cx43 accelerates transformation of cells into a NVA state or secondary necrosis and this depends on the ability of Cxs to form functional GJ channels and hemichannels.