We have applied exon amplification, GRAIL2 exon prediction and EST database searching to a 2 Mb segment of chromosome 4p16.3. Experimental and computational methods of identifying exons were comparable in efficiency and apparent false positive rate, but were complementary in gene identification, revealing distinct overlapping sets of expressed sequences. EST searching was most powerful when we considered only those ESTs that show evidence of splicing relative to the genomic sequence. The combination of the three gene finding methods produced a transcription map of 30 loci in this segment of 4p16.3 that includes known human genes, homologs of loci identified in rodents and several anonymous transcripts, including a putative novel DNA polymerase and a gene related to Drosophila ash1. While most of the genes in the region have been found, our data suggest that even with the entire DNA sequence available, complete saturation of the transcript map will require additional, focused experimental effort.