A long-range physical map of the human T-cell receptor variable alpha (TCRAV) locus was produced using 23 V alpha subgroup-specific probes. Linkage disequilibrium across the locus was also studied using polymorphic TCRAV markers. Pulsed-field gel electrophoresis was used to map V alpha gene segments onto one SfiI fragment of 500 kb and two of 200 kb using DNA from peripheral blood neutrophils. PCR and conventional Southern techniques on Jurkat, CEM, and H9 T-cell lines were used to establish the 5' to 3' order of the gene segments and the relative positions of V alpha gene segments on the SfiI fragments. The linkage disequilibrium study used single-stranded conformation polymorphism analysis to genotype 100 normal caucasoid subjects for TCRAV5S1, V6S1, V8S1, V17S1, and V21S1 polymorphisms. Strong linkage disequilibrium was detected between V5S1 and V8S1, in concordance with the physical map. This new information will be useful for future studies of genetic variation at the TCRAV locus, its role in the shaping of the TCR repertoire, and its possible contribution to autoimmune diseases.