Over the last decade, an expanding diversity of secreted phospholipases A(2) (sPLA(2)s) has been identified in mammals. Here, we report the cloning in mice of three additional sPLA(2)s called mouse group IIE (mGIIE), IIF (mGIIF), and X (mGX) sPLA(2)s, thus giving rise to eight distinct sPLA(2)s in this species. Both mGIIE and mGIIF sPLA(2)s contain the typical cysteines of group II sPLA(2)s, but have relatively low levels of identity (less than 51%) with other mouse sPLA(2)s, indicating that these enzymes are novel group II sPLA(2)s. However, a unique feature of mGIIF sPLA(2) is the presence of a C-terminal extension of 23 amino acids containing a single cysteine. mGX sPLA(2) has 72% identity with the previously cloned human group X (hGX) sPLA(2) and displays similar structural features, making it likely that mGX sPLA(2) is the ortholog of hGX sPLA(2). Genes for mGIIE and mGIIF sPLA(2)s are located on chromosome 4, and that of mGX sPLA(2) on chromosome 16. Northern and dot blot experiments with 22 tissues indicate that all eight mouse sPLA(2)s have different tissue distributions, suggesting specific functions for each. mGIIE sPLA(2) is highly expressed in uterus, and at lower levels in various other tissues. mGIIF sPLA(2) is strongly expressed during embryogenesis and in adult testis. mGX sPLA(2) is mostly expressed in adult testis and stomach. When the cDNAs for the eight mouse sPLA(2)s were transiently transfected in COS cells, sPLA(2) activity was found to accumulate in cell medium, indicating that each enzyme is secreted and catalytically active. Using COS cell medium as a source of enzymes, pH rate profile and phospholipid headgroup specificity of the novel sPLA(2)s were analyzed and compared with the other mouse sPLA(2)s.