Aims/hypothesis: The aim of this study was to estimate the heritability of quantitative measures of glucose regulation obtained from a tolbutamide-modified frequently sampled IVGTT (t-FSIGT) and to correlate the heritability of the glucose-stimulated beta cell response to the tolbutamide-induced beta cell response. In addition, single nucleotide polymorphisms (SNPs) having an exclusive effect on either glucose- or tolbutamide-stimulated insulin release were identified. Methods: Two hundred and eighty-four non-diabetic family members of patients with type 2 diabetes underwent a t-FSIGT with intravenous injection of glucose at t∈=∈0 min and tolbutamide at t∈=∈20 min. Measurements of plasma glucose, serum insulin and serum C-peptide were taken at 33 time points from fasting to 180 min. Insulin secretion rate, acute insulin response (AIR), disposition index (DI) after glucose and disposition index after tolbutamide (DIT), insulin sensitivity (SI), glucose effectiveness (SG) and beta cell responsiveness to glucose were calculated. A polygenic variance component model was used to estimate heritability, genetic correlations and associations. Results: We found high heritabilities for acute insulin secretion subsequent to glucose stimulation (AIRglucose h 2∈±∈SE: 0.88∈±∈0.14), but these were slightly lower after tolbutamide (AIRtolbutamide h 2∈±∈SE: 0.69∈±∈0.14). We also estimated the heritabilities for S I (h 2∈±∈SE: 0.26∈±∈0. 12), SG (h 2∈±∈SE: 0. 47∈±∈0.13), DI (h 2∈±∈ SE: 0.56∈±∈0.14), DIT (h 2∈±∈SE: 0.49∈±∈0.14) and beta cell responsiveness to glucose (h 2∈±∈SE: 0.66∈±∈0.12). Additionally, strong genetic correlations were found between measures of beta cell response after glucose and tolbutamide stimulation, with correlation coefficients ranging from 0.77 to 0.88. Furthermore, we identified five SNPs with an exclusive effect on either glucose-stimulated (rs5215, rs1111875, rs11920090) or tolbutamide-stimulated (rs10946398, rs864745) insulin secretion. Conclusions/interpretation: Our data demonstrate that both glucose- and tolbutamide-induced insulin secretions are highly heritable traits, which are largely under the control of the same genes.