BACKGROUND Oral clefts are common congenital anomalies and result from defects during embryogenesis. The complex etiology is evident by the number of genes and signaling pathways involved in craniofacial development. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are responsible for tissue remodeling during craniofacial development. METHODS In this study, we investigated the association of polymorphisms in 14 biologically relevant MMP and TIMP genes in 494 individuals with oral clefts and 413 control individuals from Brazil. Genotypes were generated using Taqman chemistry. Analyses were performed using PLINK software. RESULTS Polymorphisms in MMP3 (rs522616) and TIMP2 (rs8179096) showed significant association with all cleft types (all clefts, cleft lip/palate, and cleft palate; p ≤ 0.002). An additional family-based dataset (881 case-parent trios) from the United States was used for confirmation of the association findings (p < 0.05). Analysis of gene-gene interaction suggests that MMP3 and TIMP2 may interactively contribute to a cleft phenotype. CONCLUSIONS This study provides new evidence that variation in MMP3 may contribute to nonsyndromic oral clefts and further supports the involvement of TIMP2 as a cleft susceptibility gene. Although additional studies are still necessary to unveil the exact mechanism by which MMP3 and TIMP2 would contribute to a cleft phenotype, allelic polymorphisms in these genes and their interactions may partly explain the variance of individual susceptibility to oral clefts. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc.