A large number of human hematopoietic growth factor and growth factor receptor genes are localized at the long arm of chromosome 5. In this study we have determined the order of the human interleukin-3 (IL3), IL4, IL5, IL9, granulocyte macrophage-colony stimulating factor (GMCSF), and the MCSF receptor (MCSFR) genes by fluorescence in situ hybridization. Genomic lambda-clones were isolated using polymerase chain reaction (PCR)-generated probes and labeled with biotin and/or digoxigenin. These clones were first individually mapped: IL3, IL4, IL5, IL9, and GMCSF to 5q31 and MCSFR to 5q33. For ordering purposes multiple probe combinations were hybridized to metaphase chromosomes and interphase nuclei. The interphase hybridizations were evaluated by image analysis, which also allowed the measurement of the physical distance between the hybridization spots. These mapping results suggest the gene order 5cen-IL3/GMCSF-IL5-IL4-IL9-MCSFR+ ++-qter. The known genomic distance between the IL4 and IL5 genes allowed the estimation of the physical distances between the 5q31-specific genes, demonstrating that they are all within about 1.5 Mb of DNA.