Using a trichloroacetic acid precipitation procedure and gel permeation high performance liquid chromatography, we have demonstrated the presence of an antifreeze protein in the second instar larvae of the spruce budworm Choristoneura fumiferana. This protein contained a significant amount of half-cystine and only a modest content of alanine. In a radioimmunoassay, it competed with antisera against a cystine-containing antifreeze protein from sea raven, Hemitripterus americanus, thus indicating their immunological cross-reactivity and possibly, some structural homology.