Disorders of sexual development are surprisingly common. They range from mild genital abnormalities to complete sex reversal. The cause of these problems is often a failure of the complex networks of gene regulation that regulate the differentiation of testes and ovaries. There is an increasing amount of evidence that gene activity is regulated post-transcriptionally by non-coding RNAs (ncRNAs). We have performed high-throughput sequencing for small RNAs, as well as microarray analysis for long ncRNAs, for mouse embryonic gonads from 11.5 days post coitum (dpc), the time of sex determination, to 14.5 dpc, when testes and ovaries are well differentiated. A number of small and long ncRNAs were significantly differentially expressed and by using gain- and loss-of-function analysis ex vivo and in vivo we are analyzing the functional relevance of these RNAs in the developing gonads. In addition, we found that not only the expression but also the processing of microRNAs was highly controlled in a tissue-specific manner at the level of Drosha, Dicer and RISC, providing an ideal model system to identify and characterize regulatory factors.