We have demonstrated that the 5' termini of the DNAs of cauliflower mosaic virus (CaMV) isolates CM4-184 and Cabb B-JI have free hydroxyl groups accessible for phosphorylation using polynucleotide kinase. This has enabled us, using the Cabb B-JI isolate, to label and identify the 5' nucleotides. Digestion of 5' terminally labelled DNA with EcoRI restriction endonuclease revealed that only three of the eleven single-stranded EcoRI fragments were labelled, those situated adjacent to the three known gaps in the double-stranded genome. Identification of the labelled fragments indicated the polarity of the genome. Hybridization experiments using RNA transcripts from infected leaves gave results consistent with the polarity data and confirmed the direction of transcription. Transcription on the largest DNA strand (alpha strand) is initiated between map units 0.24 and 0.30 and terminates between map units 0.0 and 0.24.