We used a baculovirus vector to express the cDNA for a truncated (amino acid residues 1-848), secreted form of thyroid peroxidase (TPO) in Sf9 insect cells. Immunoreactive TPO was detected in pooled conditioned media from 10 clones using polyclonal TPO autoantibodies in the sera of patients with autoimmune thyroid disease. As a further test of TPO immunogenicity, the pooled media completely inhibited autoantibody binding to antigen. We used an ELISA to compare autoantibody reactivity to insect cell-derived TPO and TPO antigen produced by Chinese hamster ovary (CHO) cells, the standard form of antigen in present use. In a study of 22 TPO antibody-negative sera and 24 sera with different TPO autoantibody potencies, there was a highly significant correlation (r = 0.977; p < 0.001) in OD values obtained with TPO from the two different sources. The highest producing baculovirus clone generated 8.5 micrograms TPO/ml of conditioned medium, nearly 10-fold higher than previously achieved with stably transfected Chinese hamster ovary cells. Baculovirus-derived, soluble TPO therefore is an excellent source of recombinant TPO in further studies to examine the precise B cell epitopes for human autoantibodies.