Expressed sequence tags (ESTs) have been obtained from several hundred brain cDNAs as an initial effort to characterize expressed brain genes. These ESTs will become tools for human genome mapping and they will also provide candidate causative genes for inherited disorders affecting the central nervous system. We have developed a procedure for the rapid chromosomal assignment of these ESTs: cDNA sequences are first analyzed by a computer program to determine regions likely not to be interrupted by introns in the genomic DNA. A pair of oligonucleotide primers is then designed to amplify this region by the polymerase chain reaction using DNA template from human-rodent somatic cell hybrid chromosomal panels. The chromosomal assignment of the cDNA is determined by studying the segregation of the amplified products in these panels. In this paper we describe the mapping of 46 brain ESTs, as well as observations on the amplification of rodent sequences.