Previous estimates of the size of Drosophila melanogaster chromosome 4 have indicated that it is 1% to 4% of the genome or approximately 6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules of D. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from the cubitus interruptus and zfh-2 genes, which are located on chromosome 4. They each identify the same-sized distinct band that migrates at approximately 5.2 Mb in DNA preparations from the Kc cell line. We interpret this band to be intact chromosome 4. In DNA obtained from embryos of various D. melanogaster wild-type strains, this chromosome band showed strain-specific size variation that ranged from 4.5 to 5.2 Mb. The D. melanogaster chromosome 4 probes also identified a single, 2.4 Mb band in embryonic DNA from Drosophila simulans. We conclude that D. simulans chromosome 4 is substantially smaller than that of D. melanogaster, presumably owing to differences in the amount of heterochromatic DNA sequences. Our simple DNA preparation from embryos and PFGE conditions should permit preparative isolation of chromosome 4 DNA and will facilitate the molecular mapping of this chromosome.