Fluorescence in situ hybridization has been used to study the replication timing of various repeat DNA families in the short arms of human acrocentric chromosomes. In interphase nuclei, unreplicated DNA segments show singlet hybridization signals whereas replicated loci have doublet signals. The distribution of these two patterns in unsynchronized cell cultures revealed that the rRNA gene clusters replicate earlier than the closely juxtaposed alpha- and beta-satellite DNA sequences. Within the rDNA repeat unit, replication of the intergenic spacer appears to precede that of the transcribed rDNA.