The spatial and temporal relationship between the polymerase and RNase H activities of human immunodeficiency virus type 1 reverse transcriptase has been examined by using a 40-mer RNA template and a series of DNA primers of lengths ranging from 15 to 40 nucleotides, hybridized to the RNA, as substrates. The experiments were executed in the absence and presence of heparin, an efficient trap to sequester any free or dissociated reverse transcriptase, thus facilitating the study of events associated with a single turnover of the enzyme. The results indicate a spatial separation of 18 or 19 nucleotides between the two sites. To examine the effect of concomitant polymerization on the RNase H activity, the substrate was doubly 5' end labeled on the RNA and DNA. This enabled the study of RNase H activity as a function of polymerization in a single experiment, and the results in the absence and presence of heparin indicate a tight temporal coupling between the two activities.