Employing a nested polymerase chain reaction with primers from the 5' non-translated region of the enterovirus genome, we detected enteroviral RNA from patients with a variety of enterovirus-associated clinical syndromes. This technique was found to be sensitive (detecting enteroviral RNA extracted from 0.1 50% tissue culture infectious dose) and specific; no specific PCR product was detected from RNA extracts of a variety of non-enterovirus isolates. Although the technique is of comparable sensitivity to single round polymerase chain reaction followed by Southern blot hybridization, it was more rapid, since it enabled a diagnosis to be made within 1 day. Thus, using nested polymerase chain reaction we were able to detect enteroviral RNA in 31 of 46 clinical specimens from 17 of 23 patients with suspected enterovirus infections. The samples included cerebrospinal fluid, throat swabs, stool, vesicle fluid, peripheral blood lymphocytes, whole blood and pericardial effusion. In contrast virus was isolated in only 17 of 42 clinical specimens from 12 of 22 these patients. In preliminary studies, restriction endonuclease analysis of polymerase chain reaction products enabled us to distinguish between non-polio enteroviruses and poliovirus types 1, 2, and 3. This additional technique may be useful in distinguishing between such infections in polio-endemic countries where rapid public health measures may be required.