Aims/hypothesis: A genome-wide association study recently identified an association between common variants, rs1535435 and rs9494266, in the AHI1 gene and type 2 diabetes. The aim of the present study was to investigate the putative association between these polymorphisms and type 2 diabetes or type 2 diabetes-related metabolic traits in Danish individuals. Methods: The previously associated polymorphisms were genotyped in the population-based Inter99 cohort (n=6162), the Danish ADDITION study (n=8428), a population-based sample of young healthy participants (n=377) and in additional type 2 diabetes (n=2107) and glucose-tolerant participants (n=483) using Taqman allelic discrimination. The case-control study involved 4,104 type 2 diabetic patients and 5,050 glucose-tolerant control participants. Type 2 diabetes-related traits were investigated in 17,521 individuals. Results: rs1535435 and rs9494266 were not associated with type 2 diabetes. Odds ratios (OR) were ORadd 1.0 (95% C.I. 0.9-1.2; p add=0.7) and ORadd 1.1 (0.9-1.2; p add=0.4), respectively, a finding supported by meta-analyses: OR add 1.0 (0.9-1.1; p add=0.6) and ORadd 1.0 (0.9-1.1; p add=0.6), respectively. Neither rs1535435 nor rs9494266 were consistently associated with any of the tested type 2 diabetes-related metabolic traits. Conclusions/interpretation: Data from large samples of Danish individuals do not support a role for AHI1 rs1535435 nor rs9494266 as major type 2 diabetes variants. This study highlights the importance of independent and well-powered replication studies of the recent genome-wide association scans before a locus is robustly validated as being associated with type 2 diabetes.