The replication timing of a pair of natural alleles was compared at two ac-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altAl allele completes replication at between 20 and 40 min of S phase. The same region of the a4tA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both aWl and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than alt4 message on Northern (RNA) blots. The A recurring theme within the variety of recent advances in the investigation of eukaryotic DNA replication is that chromosomal context is a fundamental determinant of origin activity (20,23,44,64). It is reasonable to assume that not only origin activity but also replication fork progression and termination are sensitive to the chromosomal context. This position effect may be a reflection of the longitudinal heterogeneity of a chromosome with regard to protein composition, nucleotide base composition, transcriptional activity, higher-order structure, and association with the nuclear matrix. With this in mind, we have sought to analyze