Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type (n = 5) and heterozygous Cx 43-deficient mice (n = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 ± 5% at baseline vs. 46 ± 4 (mean ± S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 ± 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 ± 1%, P < 0.001 vs. simulated ischemia). In Cx 43-deficient cardiomyocytes, viability was also well maintained in normoxia (56 ± 10% vs. 44 ± 10%). Viability was also reduced to 17 ± 6% with 2 h simulated ischemia. In contrast to wild-type cells, preconditioning did not prevent the reduction in viability (18 ± 8%). In conclusion, Cx 43 is essential for preconditioning in the absence of gap junctions, supporting its function through improved volume regulation.