Staining of RNA with ethidium bromide (EtdBr) prior to running agarose gels has been reported to afford certain advantages over staining gels after electrophoresis. We have examined prior staining of RNA with a wide range of EtdBr concentrations, particularly with respect to its effects on Northern blot hybridizations using antisense RNA probes. Prior staining with EtdBr at concentrations of 100-1000 micrograms/ml caused significant alterations in RNA mobilities and significantly decreased hybridization with antisense RNA probes compared with unstained RNA. Prior staining with EtdBr at 10-50 micrograms/ml resulted in the best combination of staining sensitivity, absence of alterations in RNA mobility and efficiency of hybridization. Conventional staining of gels after electrophoresis also resulted in decreased hybridization efficiency with RNA probes compared with unstained RNA.