Background: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common craniofacial developmental defect. Association studies have suggested that a clefting locus is located on chromosome 6p at or near two possible loci, Factor 13A (FI3A) in the region 6p 25-24 and HLA at 6p 21.3. Aim. To test the hypothesis on the possible presence of a major gene on chromosome 6p associated with NSCLP. Patients and methods: We carried out an association study on a sample of unrelated NSCLP patients from multiplex (Mx) and simplex (Sx) families, of their unaffected relatives and in control individuals. DNA was analyzed with three PCR markers close to the putative NSCLP locus, dinucleotide repeats at loci D6S89, D6S109 and D6S105. PCR products were resolved by PAGE and visualized by silver staining. Statistical analysis was performed by means of <FONT FACE=Symbol>c</FONT>2 log ratio. Results: Significant differences were observed when comparing the allele frequency distribution of D6S89 in patients with NSCLP and controls and in patients with NSCLP-Mx and controls. No significant differences were observed for patients with NSCLP-Sx. D6S109 and D6S105 showed no significant differences in any of the comparisons. Conclusions: Our results support the hypothesis that a NSCLP locus maps on 6p23 very close to D6S89. Results for D6S109 and D6S105 do not show a clear association. Differences observed between NSCLP-MX and Sx families seem to represent different etiologic entities. The results of the present study, plus those already published for candidate loci, TGFA and MSX1, support the hypothesis that several interacting major genes participate in the etiology of NSCLP.