We have determined the chromosomal localizations of newly isolated cosmids by fluorescent in situ hybridization (FISH) on prometaphase R-banded chromosomes and have constructed a high-resolution cytogenetic map for human chromosome 8 with 416 cosmid markers, including 328 new markers and 88 reported previously. Of the 416 markers, 229 were mapped to the long arm of chromosome 8, 181 to the short arm, and 6 to the centromere. Although the clones were scattered throughout the chromosome, they were concentrated in R-positive bands. Since the estimated physical length of chromosome 8 is 135 Mb, the overall average distance between loci is 320 kb, but the average separation of loci on R-positive bands is nearly 130-200 kb. This cytogenetic map will serve as a resource for efforts to characterize chromosomal and molecular aberrations involved in cancers, to clone genes associated with hereditary diseases, and to construct a detailed physical map of large electrophoretic fragments and/or contiguous cosmids and yeast artificial chromosomes.