The posterior-vegetal cytoplasm of an ascidian egg contains maternal factors required for pattern formation and cell specification of the embryo. We report here the isolation and characterization of cDNA clones for novel maternal genes, posterior end mark 2 (pem-2), pem-4, pem-5, and pem-6. We obtained these clones from a cDNA library of Ciona savignyi fertilized egg mRNAs subtracted with gastrula mRNAs by examining the localization of the corresponding mRNAs of randomly selected clones by whole-mount in situ hybridization. As in the case of pem, all of these mRNAs were localized in the posterior-vegetal cytoplasm of the egg, and they later marked the posterior end of early embryos. The predicted amino acid sequence suggested that PEM-2 contains a signal for nuclear localization, an src homology 3 (SH3) domain, and a consensus sequence of the CDC24 family guanine nucleotide dissociation stimulators (GDSs). PEM-4 has a signal for nuclear localization and three C2H2-type zinc finger motifs, while PEM-5 and PEM-6 show no similarity to known proteins. These results provide further evidence that the ascidian egg contains maternal messages that are localized in the posterior-vegetal cytoplasm.