In Xenopus laevis there are two multigene families of 5S RNA genes: the oocyte-type 5S RNA genes which are expressed only in oocytes and the somatic-type 5S RNA genes which are expressed throughout development. The Xenopus 5S RNA replication-expression model of Gottesfeld and Bloomer (Cell 28:781-791, 1982) and Wormington et al. (Cold Spring Harbor Symp. Quant. Biol. 47:879-884, 1983) predicts that the somatic-type 5S RNA genes replicate earlier in the cell cycle than do the oocyte-type genes. Hence, the somatic-type 5S RNA genes have a competitive advantage in binding the transcription factor TFIIIA in somatic cells and are thereby expressed to the exclusion of the oocyte-type genes. To test the replication-expression model, we determined the order of replication of the oocyte- and somatic-type 5S RNA genes. Xenopus cells were labeled with bromodeoxyuridine, stained for DNA content, and then sorted into fractions of S phase by using a fluorescence-activated cell sorter. The newly replicated DNA containing bromodeoxyuridine was separated from the lighter, unreplicated DNA by equilibrium centrifugation and was hybridized with DNA probes specific for the oocyte- and somatic-type 5S RNA genes. In this way we found that the somatic-type 5S RNA genes replicate early in S phase, whereas the oocyte-type 5S RNA genes replicate late in S phase, demonstrating a key aspect of the replication-expression model.