Since the discovery of RNAi small RNA molecules have been under intense study. They have been shown to impact many different processes, ranging from development to organ function and carcinogenesis. Recently, it has become clear that many distinct small RNA families exist. However, all act through a member of the well-conserved Argonaute family of proteins. I try to understand how specificity of the different Argonaute proteins is achieved, and I am particularly interested in Argonautes that may contribute to the epigenetic marking of genomic DNA in animals. My focus is on Argonaute function in the vertebrate germline, a tissue that is an especially intriguing system with regard to the resetting and establishment of epigenetic marks. As model system I use the zebrafish. Piwi proteins are Argonaute proteins that in vertebrates are specifically expressed in germ cells, and have been implicated in modifying chromatin structures. We demonstrated that zebrafish piwi is expressed in both the male and the female gonad and that loss of piwi results in loss of germ cells due to apoptosis. We have characterized small RNAs that bind to piwi (piRNAs) in both ovary and testis, and found that they play a role in the silencing of transposable elements. Furthermore, we have shown that the biogenesis of piRNAs differs markedly from that of other small RNAs like miRNAs. The experiments I propose address how Piwi proteins and piRNAs act in germ cells to ensure a functional germline and a stable propagation of intact chromatin over generations. First, I will address the biogenesis of piRNAs. Second, I will identify novel components of the Piwi pathway. Third, I will address the mode(s) of action of piRNAs. On all fronts a combination of genetics, molecular biology and biochemistry will be used.