Homogenates, total particulate and plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [3H]LY181984 with high affinity (Kd of 20 to 50 nM). Highest affinity binding (Kd of 25 nM) was to purified plasma membrane. The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance constituent representing about 1/1000 of the total plasma membrane proteins. When corrected for mitochondrial uptake, binding recoveries of about 80% were achieved. Of the recovered specific radioactivity bound, approximately 90% was associated with the total particulate fraction. Of this, nuclei- and plasma membrane-free total membranes bound little or no [3H]LY181984 with high affinity. The high-affinity binding was restricted primarily to the plasma membranes. All fractions exhibited varying degrees of lower affinity binding indicative of a heterogeneous array of components capable of binding [3H]LY181984 at high concentrations of LY181984. Enrichment of 5-fold over total homogenates of high-affinity binding compared favorably to a 6.7-fold enrichment of the plasma membrane marker enzyme 5'-nucleotidase determined in parallel. We conclude that plasma membranes of HeLa cells contain high-affinity binding sites of low abundance for the antitumor sulfonylurea LY181984 and that the high-affinity sites are associated predominantly with the plasma membrane.