The impact of genetic factors and maternal diabetes on glucose tolerance and pancreatic β-cell function was studied in first generation (F1) offspring generated in crosses between the spontaneously diabetic Goto-Kakizaki (GK)-Wistar rat and normoglycemic control Wistar rats (W). The (GK × W) F1 hybrids were offspring of either male GK (mGK) and female Wistar (fW) (mGK × fW) or male Wistar (mW) and female GK (fGK) (mW × fGK) rats. Already at 8 days of age, blood glucose levels were elevated in GK (7.6 ± 0.5 vs. 4.8 ± 0.3 mM in W; P < 0.001) and in F1 rats (6.0 ± 0.3 in mGK × fW and 6.6 ± 0.4 mM in mW × fGK; both P < 0.01 vs. W). In 2-month-old male rats, glucose (2 g/kg, intraperitoneally) markedly increased blood glucose levels after 60 min in GK rats (18.1 ± 0.6 vs. 5.5 ± 0.3 mM in W; P < 0.001) and moderately increased levels in F1 rats (9.9 ± 0.9 in mGK × fW and 11.6 ± 1.0 mM in mW × fGK, both P < 0.01 vs. W). Similar patterns were obtained in female rats. Repeated backcrossing of F1 with W rats successively improved glucose tolerance. In perfused pancreases of male rats, the 20-min insulin response to 16.7 mM glucose was −7.44 ± 5.18 pmol in GK rats, 71.57 ± 12.25 pmol in W rats, 9.00 ± 0.89 pmol in mGK × fW rats, and 18.20 ± 3.97 pmol in mW × fGK rats. In female W rats, the glucose-induced insulin response was significantly lower than in males (P < 0.05). However, as in males, insulin responses to glucose were impaired in both GK and F1 female rats. Arginine-induced insulin release was similar in all groups. In mGK × fW, glucose-stimulated somatostatin release was 50% of that in W rats, whereas arginine-stimulated responses of glucagon and somatostatin were not different from W rats. Pancreatic contents of insulin and glucagon were similar in mGK × fW and W rats, whereas somatostatin content was lower in mGK × fW rats (P < 0.05). In conclusion, the diabetic state in GK and F1 rats was evident early in life. Hybrid rats were intermediate between W and GK rats with regard to glucose tolerance and glucose-stimulated insulin response in vitro, but had normal pancreatic insulin content. Results of repeated backcrossing of F1 rats with W rats showed that genes in >1 locus contribute to the diabetic state. Furthermore, the absence of significant differences between impact of maternal and paternal origin of the GK genes for glucose intolerance suggests that hyperglycemia in utero does not influence the severity of diabetes in the F1 offspring.