A longstanding hypothesis proposes that amyotrophic lateral sclerosis-motor neurone disease (ALS-MND) is a late consequence of subclinical poliovirus (PV) infection. In this study, RNA extracts of CNS tissue from 28 patients with ALS-MND and 7 controls were assayed by nested polymerase chain reaction (PCR) using primers to the 5'-untranslated region (UTR) of the enterovirus (EV) genome which is highly conserved between EVs including PV, echovirus and coxsackie viruses. The integrity of RNA extracted from either archival paraffin-embedded or frozen CNS tissue was assessed by detection of constitutive Ableson tyrosine kinase (ABL) mRNA by PCR. Of 63 tissue samples assayed, 81% (51/63) were ABL-positive corresponding to 78% (22/28) of the ALS-MND cases and all controls, None of the 27 ALS-MND cases (i.e, 21 ABL(+) and 6 ABL(-)) in which paraffin-embedded tissue was used nor any of the age and sex matched controls were positive for specific PV/EV RNA. Moreover, CNS tissue from 14 different locations obtained from one patient <2 hrs after death and immediately frozen, showed no evidence of PV/EV at any site by PCR. Disease duration, degree of tissue autolysis and duration of tissue storage were all excluded as factors which may predispose to negative results. The sensitivity of the PV PCR was determined to be 40-400 copies (12.5-125 ag) of synthetic EV RNA transcripts in i mu g of cellular RNA and the assay was shown to detect all types of PV and other EVs tested. Thus it seems unlikely that a persistent PV or related EV infection is implicated in ALS-MND unless there has been alteration in the 5'-UTR of the virus genome.