The neurexin-1 gene (NRXN1) has been shown to play a fundamental role in synaptogenesis and synaptic maintenance, as well as Ca(2+) channel and NMDA receptor recruitment. A recent study reported that NRXN1 is associated with nicotine dependence (ND); this, together with the intriguing physiological functions of the gene, motivated us to investigate the involvement of NRXN1 with ND in independent samples. In this study, we analyzed 21 single nucleotide polymorphisms (SNPs) within NRXN1 for association with ND, which was assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerström test for ND (FTND). Individual SNP and haplotype association tests were carried out in a sample consisting of 2037 individuals from 602 nuclear families of African-American (AA) or European-American (EA) origin. Individual SNP analysis revealed significant associations of rs2193225 with SQ, HSI and FTND (P = 0.00014-0.0010) in the EA sample and with SQ (P = 0.0019) in the pooled sample under the dominant model and rs6721498 with SQ, HSI and FTND in the AA (P = 0.000090-0.0000086) and pooled (P = 0.0010-0.00099) samples under the additive model, following correction for multiple testing. Haplotype analysis revealed six major haplotypes in the AA sample (minimum P-value = 0.000079), one major haplotype in the EA sample (P = 0.0062) and five major haplotypes in the pooled sample (minimum P-value = 0.00083), which showed significant association with all three ND measures; all of these contained one specific allele from one of the two aforementioned SNPs. Based on our findings that NRXN1 has significant association with ND in two independent samples, recent findings that NRXN1 plays an important role in synaptic development, and the previous report of association, we conclude that this gene represents a strong candidate for involvement in the etiology of ND.