In this report we use several previously described methods, in novel combination, to establish a sensitive and flexible nonradioactive method. First, we prepared single-stranded digoxigenin-labeled probes using a high-efficiency polymerase chain reaction (PCR) method (4). For detection of RNA, blots were hybridized with probes containing digoxigenin and then incubated with alkaline phosphatase-conjugated anti-digoxigenin antibody. Bound probe was rapidly detected with X-ray film using localized light emission from the reaction of alkaline phosphatase with lumigen-paraphenylenediamine substrate (5). This method allows flexibility in probe sequence selection, independent of restriction enzyme site location, and it works well with small probes. This approach allows sensitive differential analysis of closely related members of a gene family.