Significant association between polymorphisms at the ANK3 gene with bipolar disorder has previously been reported and confirmed in several samples. Here we report on association between ANK3 and bipolar disorder in a new sample of 593 patients and 642 controls (UCL2) as well as the results of sequencing of the exons and flanking regions of ANK3 from bipolar patients. Single nucleotide polymorphisms (SNPs) associated with bipolar disorder in our original GWA study (UCL1) were genotyped and tested for association in the new sample. Novel SNPs found by sequencing were genotyped in both samples to test for association with bipolar disorder. None of the SNPs previously associated with bipolar disorder were associated in the UCL2 sample. One of the four SNPs associated in the UCL1 sample, rs1938526, was still significantly associated with bipolar disorder when the UCL1 and UCL2 samples were combined (P = 0.0095). The results demonstrate the impact of heterogeneity on replication of allelic associations even within well-defined ancestral populations. DNA sequencing revealed a novel low frequency (0.007) ANK3 SNP (ss469104599) which causes a non-conservative amino acid change at position 794 in the shorter isoforms of the ankyrin G protein. Protein-function analysis software predicted the amino acid change to be “probably damaging” and it could therefore be detrimental to the function of this isoform. Given that there was only a modest increase in the allele frequency of ss469104599 in cases compared to controls further association studies are needed in additional samples to establish a possible etiological role for this amino acid change. © 2012 Wiley Periodicals, Inc.