A study was undertaken to enumerate and partially characterize soluble factors generated by tumor-bearing animals capable of suppressing PHA-induced splenocyte proliferation. Sprague-Dawley rats were induced to form malignant ascites by the intraperitoneal injection of the Walker 256 carcinoma. Intact ascites suppressed splenocyte proliferation by 96%. Molecular sieving of the ascites by means of ultrafiltration (10-kilodalton particle cutoff) revealed suppressor activity to reside in both the ultrafiltrate and retentate. Further enumeration of suppressor factors was achieved by preparative polyacrylamide gel electrophoresis of the ascites ultrafiltrate and retentate. Five discrete bands of suppressor activity were resolved in the ultrafiltrate, three of which were heat-labile. Three discrete bands of suppressor activity were resolved in the retentate, none of which were heat-labile. This study underscores the complexity and heterogeneity of soluble factors elaborated in a cancer-bearing animal.