The serological relationship of herpes simplex type 1 virus and Herpesvirus simiae was studied. Antibody titers to these viruses were determined in 163 Macaca mulatta sera and 67 human sera by serum neutralization (SN) and complement fixation (CF) tests. Both groups of sera were also tested by CF with envelope and capsid antigens of herpes simplex type 1. By SN, the majority of the monkeys and all of the humans had a higher titer to herpes simplex type 1 than to H. simiae. By CF, with crude antigens the titers in the monkey sera were greater to H. simiae than to herpes simplex type 1, although four sera had equal titers to both antigens; the titers in the human sera were conversely higher with the herpes simplex type 1 antigen, except for four sera which had equal titers to both antigens. The capsid CF antigen of herpes simplex type 1 was reactive with the human sera but virtually nonreactive with the monkey sera; the envelope CF antigen of herpes simplex type 1 was reactive with both monkey and human sera but was somewhat less reactive than the crude herpes simplex type 1 CF antigen. In addition, serum samples from a patient recently infected with H. simiae were examined by CF and SN for antibody to both herpes simplex type 1 and H. simiae viruses. The serological profile indicated a positive correlation with the infecting virus. Although the SN titers did not conclusively reflect an infection with H. simiae, the CF titers were higher to H. simiae than to herpes simplex in later sera and thus appeared to be compatible with H. simiae infection.