Our previous studies have suggested that elevated lactogen, increased glucose-stimulated insulin secretion, and increased β-cell coupling are associated. To determine whether this association occurs under conditions of physiologically increased lactogen, we have studied the extent of dye coupling in rat islets during the later stage of pregnancy. These animals have high plasma lactogen levels in the form of placental lactogen, increased plasma insulin, and decreased plasma glucose. The fluorescent tracer, Lucifer yellow CH, was microinjected into central cells of islets from both pregnant and virgin rats, and the extent of transfer was quantitated by determining the projected area of dye spread. Two area measurements were made for each injection, one around the entire discernible fluorescent region ('outer') and another around the distinct brighter region of cells surrounding the injected cell ('inner'). Pregnancy increased dye transfer, as determined by both measurements. The outer area of dye transfer was 9047 ± 775 μm2 for the islets from pregnant rats and 4699 ± 391 μm2 for the islets from virgin rats (P<.001). Similarly, pregnancy increased the inner area of dye transfer, 1447 ± 161 μm2 for the islets from pregnant rats and 795 ± 80 μm2 for the islets from virgin rats (P<.001). These results support the hypothesis that elevated lactogen, increased glucose-stimulated insulin secretion, and increased β-cell dye coupling are associated under physiological conditions. The study indicates that enhanced β-cell coupling is part of the structure and functional adaptation that the islets undergo during a subject's pregnancy and demonstrates that the extent of β-cell coupling is regulated by a physiological condition.