Radiation hybrid mapping was used in conjunction with a natural deletion mapping panel to predict the order of and distance between 13 loci in the distal portion of the long arm of human chromosome 5. A panel of irradiation hybrids containing fragments of 5q was generated from an HPRT+ Chinese hamster-human cell hybrid containing a derivative chromosome 5 [der(5)t(4;5)(5qter----5p15.1::4p15.1----4pter)] as its only human DNA. One hundred nine radiation hybrids containing human DNA were screened with polymerase chain reaction primer sets representing nine genes encoding growth factors, growth factor receptors, or hormone receptors (IL3, IL4, IL5, CSF1R, FGFA, ADRB2, GRL, GABRA1, and DRD1) as well as four other loci (FER, SPARC, RPS14, and CD14) to generate a radiation hybrid map of the area 5q21-q35. A physical map predicting the order of and distance between the 13 loci was constructed based on segregation of the 13 loci in hybrid clones. The radiation hybrid panel will be useful as a mapping tool for determining the location and order of other genes and polymorphic loci in this region as well as for generating new DNA probes from specific regions.