An improved chemiluminescence-based RNA/DNA detection procedure offering a widely applicable alternative to the conventional 32P labeling employed in molecular biology is described. Even highly sensitive applications such as Northern blot analysis of low-copy RNAs are shown to be feasible now without radioactive labeling. Improved quality of nonradioactive detection was obtained by the use of digoxigenin-labeled nucleotides in combination with dioxetane substrates which are decomposed by the hydrolysis of alkaline phosphatase. Previously existing problems involving unacceptably high background signals in nonradioactive labeling procedures were eliminated by the application of a modified RNA/DNA transfer, hybridization, and detection protocol. The data presented here delineate a system consistently superior to radioactivity and should considerably increase the usefulness of nonradioactively labeled probes detected by chemiluminescence.