Human replication protein A (RP-A) is a three-subunit protein that is required for simian virus 40 (SV40) replication in vitro. The trypanosome homologue of RP-A has been purified from Crithidia fasciculata. It is a 1:1:1 complex of three polypeptides of 51, 28, and 14 kDa, binds single-stranded DNA via the large subunit, and is localized within the nucleus. C. fasciculata RP-A substitutes for human RP-A in the large tumor antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase alpha/primase, but it does not support efficient SV40 DNA replication in vitro. This extraordinary conservation of structure and function between human and trypanosome RP-A suggests that the mechanism of DNA replication, at both the initiation and the elongation level, is conserved in organisms that diverged from the main eukaryotic lineage very early in evolution.