Isolation of the virus, the most reliable means of diagnosing Colorado tick fever, is not always practicable. The standard complement-fixation test, using a mouse-brain-derived antigen, may not detect significant rises in antibody titer during the usual 2- or 3-week observation period. Complement-fixation (CF) tests, with antigens derived from infected mouse brains and from an established line of hamster-kidney cells, were compared with a neutralization (plaque-reduction) test and an indirect fluorescent-antibody (IFA) staining test for usefulness in the serologic diagnosis of 34 cases of Colorado tick fever. The cell-culture antigen was as reliable as the mouse-brain antigen, and its preparation was simpler. The CF-antibody titers and the neutralizing-antibody titers tended to be lower and slower to appear than the IFA titers. The IFA method was simple, rapid, and accurate. The IFA titers frequently appeared earlier after onset of illness and reached higher levels than did the CF titers.