We have investigated the role of gap junctions in Caenorhabditis elegans oocyte maturation and fertilization. Gap junctions are observed between oocytes and the surrounding ovarian sheath cells in wild-type gonads. The sheath transcription factor CEH-18 is required to negatively regulate oocyte maturation, mitogen-activated protein kinase (MAPK) activation, and ovulation. Transmission electron microscopy (TEM) indicates that sheath/oocyte gap junctions are rare or absent in ceh-18(mg57) null mutant gonads. To test the hypothesis that gap junctions negatively regulate oocyte maturation, we performed an RNAi screen of innexin genes, which encode channel-forming proteins. Here we show that INX-14 and INX-22 are required in the female germ line to inhibit oocyte maturation, MAPK activation, and ovulation. Genetic analysis and TEM are consistent with INX-14 and INX-22 being components of sheath/oocyte gap junctions. Our results support the hypothesis that gap junctions maintain oocytes in meiotic prophase I when sperm are absent. We also implicate these channels in regulating sheath cell contractile activity and sperm recruitment to the spermatheca, the site of sperm storage and fertilization. Together with previous studies, our results help establish the C. elegans gonad as a model system for investigating the molecular mechanism(s) by which gap junctions regulate meiosis and fertilization. © 2006 Elsevier Inc. All rights reserved.