Wolf-Hirschhorn syndrome (WHS) is a multiple malformation syndrome characterised by mental and developmental defects resulting from the absence of a segment of one chromosome 4 short arm (4p16.3). Due to the complex and variable expression of this disorder, it is thought that the WHS is a contiguous gene syndrome with an undefined number of genes contributing to the phenotype. In an effort to identify genes that contribute to human development and whose absence results in this syndrome, we have utilised a series of landmark cosmids to characterise a collection of WHS patient derived cell lines. Fluorescence in situ hybridisation with these cosmids was used to refine the WHS critical region (WHSCR) to 260 kb. The genomic sequence of this region is available and analysis of this sequence through BLAST detected several cDNA clones in the dbEST data base. A total of nine independent cDNAs, and their predicted translation products, from this analysis show no significant similarity to members of DNA or protein databases. Furthermore, these genes have been localised within the WHS critical region and reveal an interesting pattern of transcriptional organisation. A previously published report of a patient with proximal 4p- syndrome further refines the WHSCR to 165 kb defined by the loci D4S166 and D4S3327. This work provides the starting point to understand how multiple genes or other mechanisms can contribute to the complex phenotype associated with the Wolf-Hirschhorn syndrome.