Studies were performed to determine if periovulatory ovine follicles secrete chemoattractants for leukocytes, and if so, to begin to elucidate the chemical nature of such factors. Tissues were obtained at 0, 12, 24, and 36 h after initiation of the preovulatory surge of luteinizing hormone and placed in short-term incubation (ovulation occurs at approximately 24 h). Follicular-conditioned medium was tested for its ability to attract leukocytes by utilizing a linear under-agarose assay: chemotaxis was quantified as a function of the leading front of migration of cells. Neutrophils and eosinophils were attracted toward media conditioned with tissues of 24 and 36 h. Monocytes responded toward medium of tissues collected at 36 h. There was no evidence for chemoattraction of basophils or lymphocytes. Chemoattractant activity for granulocytes and monocytes was of low molecular weight origin (less than 3000) and water-soluble. High-performance liquid chromatographic separation of this sample produced a distinct peak with recoverable activity. The isolated fraction was rich in glycine. Eosinophils also migrated toward an additional low molecular weight attractant that was extracted into ethyl acetate. Leukocytes attracted into periovulatory follicles might produce substances (eg., proteolytic enzymes and angiogenic factors) that play a role in the mechanisms of ovulation and luteinization.