Colorado tick fever (CTF) virus elicits an acute illness in humans, producing nonspecific flu-like symptoms and a biphasic fever in approximately 50% of patients. The disease is transmitted by the adult Rocky Mountain wood tick (Dermacentor andersoni), and therefore incidence is limited by the habitat and life cycle of that vector. The early symptoms of infection are difficult to distinguish from those of several other agents, especially Rickettsia rickettsii. Serologic testing is usually unable to provide evidence of CTF viral infection during the acute phase because of the late appearance of the various antibodies. Here we report the development and clinical application of a test to diagnose this disease during the acute stages. Oligonucleotide primers to the S2 segment of CTF (Florio) virus were made, and these were used in the amplification of a 528-bp fragment of DNA, transcribed from the double-stranded CTF virus RNA template by reverse transcriptase PCR. RNAs processed from 16 CTF virus isolates yielded similar results when analyzed on agarose gels. These were distinguishable from their antigenic relatives Eyach, S6-14-03, and T5-2092 and from other coltiviruses and an orbivirus but not from the antigenically distinct CTF virus-related isolate 720896. A mouse model demonstrated the utility of this method with whole-blood specimens, and CTF virus was successfully detected in human sera from the initial day of the onset of symptoms to 8 days later. The reverse transcriptase PCR method is a promising tool for the early diagnosis of CTF viral infection, or for ruling out CTF virus as the etiologic agent, in order to facilitate appropriate medical support.