Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.