We isolated a panel of 20 DNA probes that hybridize specifically to chromosome No. 4 of Plasmodium falciparum and used them to construct a restriction map of chromosome No. 4 in the FCR3 strain. These probes were partially sequenced and had an insert size range of 70-310 bp (average 160 bp) and a GC content range of 19-53% (average 35%). Three probes were identical to previously described P. falciparum sequences. Two probes were homologous to an 18-bp repetitive sequence in a previously cloned P. falciparum gene but were not homologous to other parts of the gene. One probe sequence is a homologue of the heat shock protein, DnaJ. The location of these probes and four previously cloned probes on chromosome No. 4 were determined by using eight restriction enzymes that recognize 6-bp sites containing only G or C and 10 restriction enzymes that recognize 6-bp sites containing four G or C and two A or T. The locations of the probes were well distributed along the chromosome. Three probes were located at two sites and two probes were found at at least four sites on chromosome No. 4. Maps of chromosome No. 4 of the FCR3 strain, and three laboratory-selected, pyrimethamine-resistant derivative strains were constructed. Two of the strains, FCR3-D81 and FCR3-D85, each had two polymorphic forms of chromosome No. 4. Each of those polymorphic chromosomes had a duplicated part of the center of the chromosome making the cell diploid for the genetic material in that region. Those chromosomes also had an amplification and probable intrachromosomal translocation of a 50-kb fragment of chromosome No. 4. One strain derived from FCR3-D7, FCR3-D7-1 contained 20 copies of a tandemly amplified fragment carrying the dihydrofolate reductase-thymidylate synthase gene and an amplification of an unrelated part of the chromosome.