Single-stranded RNA probes were developed from cloned cDNA fragments derived from four picornaviruses; poliovirus type 1, coxsackievirus B3, ECHOvirus 9 and Theiler's murine encephalomyelitis virus. In comparative testing with a nick-translated cDNA probe, the RNA probe was found to be 10-100-fold more sensitive. Hybridization conditions were optimized for RNA probes to picornavirus targets. Longer hybridization times gave an improved signal, as did a temperature of 50 degrees C. Formamide concentration had little effect on signal strength. A crude transcription mix performed as well or better as a probe than did more purified preparations of RNA. Finally, a combination of the three probes derived from human picornaviruses detected all 16 serotypes of human enteroviruses tested. Used individually, the four probes detected different spectra of animal and human picornaviruses, shedding more light on the interrelationships among these pathogens.