In an attempt to identify trans-acting factors involved in replication origin function, we have characterized the RAR3 and RAR5 genes, identified by mutations which increase the mitotic stability of artificial chromosomes whose replication is dependent on the activity of weak ARS elements. Sequence analysis has shown that the RAR3 gene is identical to GAL11/SPT13, which encodes a putative transcription factor involved in the expression of a wide range of genes. Change-of-function mutations that truncate the RAR3 protein appear to be required to enhance chromosome stability. In contrast, loss of the RAR5 protein results in enhanced chromosome stability, as if the protein is an inhibitor of ARS function. The RAR5 gene encodes the 175 kDa DNA strand transfer protein beta, an activity that can promote the transfer of a strand from a double-stranded DNA molecule to a complementary single strand. This observation implies that a presumed recombination activity can affect eukaryotic chromosomal replication.