A proline rich antigen (PRA), which protects mice against Coccidioides immitis, has been analyzed for differential expression and variation among isolates. Northern blots of RNA from different stages of growth were probed with previously cloned cDNA and showed that mRNA for PRA increased as spherules transformed and matured from mycelia. This pattern corresponds to the relative potency of whole cell vaccines from similar preparations. The PRA gene was then cloned from a genomic library of the Silveira strain of C. immitis and its sequence analyzed. Eight other coccidioidal isolates, selected for diversity in geographic origin and resulting clinical disease, demonstrated heterogeneity in Southern blots and in sequences of polymerase chain reaction products. Silveira differed from other California isolates at 33 of 555 bases, whereas it differed from non-California isolates by <=2 bases. Only one of these substitutions affected protein sequence. Thus, tests or vaccines based on this gene are likely to cover most isolates.