To delineate potential structural mechanisms of impaired cell coupling induced by hypoxia in canine myocardium, we characterized derangements in intercellular junctions and alterations in the space constant in strips of ventricular epimyocardium before and after selected intervals of hypoxia in vitro. Tissue samples were analyzed morphometrically with transmission and freeze-fracture electron microscopy. Space constants in control tissues averaged 1.61 +/- 0.47 mm (mean +/- SD). After 30 and 60 min of hypoxia, space constants declined by 0.24 +/- 0.22 and 0.32 +/- 0.17 mm, respectively (P less than 0.05 vs. control in each case). Impaired coupling was not reversible with reoxygenation. Focal pathological separation of intercalated disk membranes was observed after 30 min of hypoxia, but morphometric analysis demonstrated no reduction in gap junction surface density to account for uncoupling after 30 min of hypoxia. However, after 60 min of hypoxia, gap junction surface density was reduced by 45%. Quantitative analysis of freeze-fractured gap junction replicas after 30 min of hypoxia revealed a significant decrease in P-face particle diameter from 8.51 +/- 1.64 nm in control tissues to 7.25 +/- 1.33 nm (P less than 0.01) with no further change at 60 min. Thus impaired coupling at 30 min is likely related to a change in the gap junction particle. Further uncoupling after 60 min of hypoxia is likely related, in addition, to reduced gap junction surface density. These results suggest that alterations in P-face particles and gap junction surface density are important determinants of progressive cellular uncoupling induced by hypoxia.