We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.The replication of eucaryotic chromosomes is under both spatial and temporal controls, as evidenced by the firing of local clusters of replicons according to developmentally regulated programs and the replication of particular chromosome domains during similar intervals of the S phase in successive cell cycles (reviewed in reference 16). That the genomic location of an immunoglobulin gene sequence can influence its schedule of replication has been shown by Calza et al. (3).A relationship between transcriptional activity and the timing of replication has been inferred based on the observation that genes are generally replicated earlier in the S phase in cells where they are transcribed than in cells where they are inactive (7, 13). In the case of the Xenopus laevis kidney cell line TrXo, translocation of oocyte-type 5S rDNA genes from their normal telomeric location to a pericentromeric location is correlated with both a shift from late to early S-phase replication and the accumulation of oocyte 5S transcripts (11, 15), supporting the suggestion that the earlier replication of somatic versus oocyte 5S genes in normal cells may contribute to the selective transcription of the somatic genes (14,38).A structural link between replication and transcription is suggested by the observations that under normal cell growth conditions the Hi histones and octamer cores of bulk chromatin are conservatively replicated (23-25, 34, 36), whereas the core histone octamers of transcribed chromatin and the HMG 14/17 nonhistone pair preferentially associated with transcribed...