The human type 2 cystatins are encoded by a multigene family of eight to nine members. Five functional genes (CST1-CST5) and two pseudogenes (CSTP1 and CSTP2) have previously been cloned. We have developed a panel of short genomic fragments that serve as gene-specific probes for these seven genes under hybridization conditions used for Southern blots or fluorescence in situ hybridization (FISH). Analysis of BssHII-digested genomic DNA using these probes was consistent with association of all seven genes with a 905-kb fragment, suggesting physical clustering at a single site. The cluster was assigned to chromosome 20 by screening a somatic cell hybrid panel with an STS for CST2. This assignment was confirmed for each gene by FISH using repeat-free gene-specific genomic probes ranging in size from 2.3 to 0.8 kb. All the loci mapped to 20p11.2.