We analyzed DNA restriction fragment length polymorphism (RFLP) in 53 white ankylosing spondylitis (AS) patients and 92 healthy controls, utilizing a full-length HLA-B7 complementary DNA probe. A 9.2-kilobase (kb) Pvu II fragment was found to be significantly increased in frequency in B27 positive AS patients compared with the B27 positive control group (P = 0.00085, relative risk = 7.2). The presence of both B27 and the 9.2-kb RFLP in an individual conferred a relative risk for AS of 297, compared with a relative risk of 119 in those who had B27 alone. The 9.2-kb RFLP appears to be a marker for AS which, with HLA-B27, contributes further to susceptibility to the disease. Our findings indicate that this fragment and HLA-B27 segregate independently in familial studies of AS.