Marfan syndrome (MFS), a common connective tissue disorder, is caused by fibrillin-1 (FBN1) mutations that are scattered throughout the gene and are largely unique to individual families. Mutation detection in this large gene of 65 exons is a considerable technical challenge. To develop an efficient method capable of identifying all possible mutations in this gene, we have explored the use of a novel denaturing high-performance liquid chromatography (DHPLC) system. This technique compares two or more chromosomes as a mixture of denatured and reannealed PCR amplicons. Under partially denaturing conditions, heteroduplexes can be separated from homoduplexes. A panel of 94 DNA samples from individuals with MFS or related connective tissue disorders was screened exon-by-exon by this method. A total of 66 unique heteroduplex profiles was identified. Sequencing of the amplicons detected 37 novel and two previously reported mutations, as well as 15 novel and 10 known polymorphisms or unique sequence variants that are probably of no clinical significance. Of the 34 mutations found in definitive MFS cases, 16 were identified in the 21 samples that had not been screened before (76% detection rate) and 17/40 (43%) were in samples previously screened by other mutation detection methods. In 32 individuals with MFS-related phenotypes, five FBN1 mutations were identified (16%). Our results demonstrate the power of the DHPLC method to detect FBN1 mutations. It should be applicable for mutation screening in any gene in a large population.