Replication fork polarity methods measure the direction of DNA synthesis by taking advantage of the asymmetric nature of DNA replication. One procedure that has been used on a variety of cell lines from different metazoans relies on the isolation of newly replicated DNA strands in the presence of the protein synthesis inhibitor emetine. Since Okazaki fragments are not synthesized under such conditions, DNA strands produced during continuous exposure to emetine consist mainly of leading strands. In the protocol described, leading strands isolated from emetine treated cells are detected with single-stranded probes representing each strand of the DNA duplex in the region of interest. Hybridization of leading strands to one strand of a cloned genomic template identifies the direction of replication fork movement. If initiation of DNA synthesis occurs from a preferred site, leading strands should diverge from the corresponding initiation region. The leading strand method is particularly useful for mapping initiation in chromosomal loci that do not replicate immediately on entry into S phase and in mapping the replication fork patterns in which candidate initiation regions have not been identified. Cautious interpretation of the results is needed because the method relies heavily on quantitative hybridization. Leading strand data can be difficult to interpret when the genomic targets are very close to initiation regions and when the targets vary in their hybridization efficiency or in the efficiency of incorporation of nucleotide analogs. The experimental details of the method are reviewed, controls to avoid common pitfalls are suggested, protocols to facilitate the accurate interpretation of the results are provided.