Heat stress (HS) induces hyperinsulinemia and hypoglycemia in lactating dairy cows. We hypothesized that γ-aminobutyric acid (GABA) participates in the regulation of this altered glycemic state as it is produced by the pancreatic beta cells and has a stimulatory effect on pancreatic secretion of insulin. Multiparous lactating Holstein cows (n=6; 63.33±2.35 DIM, 3.17±0.40 lactations) were placed in environmentally controlled rooms for four experimental periods: 1) thermoneutral (TN; d 1-5; 18±4°C), 2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; d 6-10), 3) heat stress (HS; d 16-20; 33±4°C), and 4) HS + euglycemic clamp (EC; d 21-25). Cows were milked twice daily, and blood samples were collected once daily via coccygeal venipuncture into heparinized evacuated tubes. Plasma GABA concentrations were determined using a competitive ELISA. The data were analyzed in two ways. The first analysis included data from all treatment periods and yielded no period-based differences in plasma GABA concentrations. In this analysis, plasma GABA was lowly correlated to plasma insulin concentrations (r = -0.29, P