cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5' --> 3' exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.