Sera from cancer patients and healthy individuals, obtained from two independent sources, were examined for their abilities to react with herpes simplex virus-associated tumor antigens, AG-4 and NVA-TAA (nonvirion antigen-tumor-associated antigen). Both antigens were prepared by infection of HEp-2 cells with herpes simplex virus type 2, and all antigen-antibody interactions were measured by the micro-complement fixation test. Of sera from 16 patients with cancer of the uterine cervix, 81% (P less than 0.01) reacted with NVA-TAA, whereas 78% (P less than 0.001) of 18 sera examined reacted with AG-4. These values differed significantly from those for normal sera, of which 14% reacted with NVA-TAA and 13% with AG-4. Of sera for 8 patients with squamous cell carcinoma of head and neck or vulva, 75% (P less than 0.02) reacted with NVA-TAA, whereas 63% (P less than 0.05) reacted with AG-4. As a group, other cancers (including adenocarcinoma of lung, breast, ovary, and cervix; liposarcoma; sarcoma; melanoma; and carcinoma of the endometrium) did not differ significantly from controls in reactive patterns with AG-4 or NVA-TAA. These studies partly supported the reported preferential reactivity of AG-4 and NVA-TAA with sera of patients with squamous cell carcinoma, especially of the uterine cervix.