Summary Myotubularins (MTMs) constitute a large family of lipid phosphatases that specifically dephosphorylate phosphatidylinositol (PI) (3)P. MTM1 and MTM2 are mutated in X-linked myotubular myopathy and Charcot-Marie-Tooth disease (type 4B) respectively, although.the mechanisms whereby MTM dysfunction leads to these diseases is unknown. To gain insight into MTM function, we undertook the study of MTMs in the nematode C. elegans, which possess a representative homologue of the four major subgroups of MTMs identified in mammals (vertebrates). As in mammals, we found that C. elegans MTMs mediate distinct functions. let-512 (vps34) encodes the C. elegans homolog of the yeast and mammalian homolog of the phosphatidylinositol 3-kinase Vps34. We found that reduction of mtm-6 (F53A2.8) function by RNA inhibition (RNAi) rescued the larval lethality of let-512 (vps34) mutants and that the reduction of mtm-1 (Y110A7A.5) activity by RNAi rescued the endocytosis defect of let-512 animals. Together, these observations provide genetic evidence that MTMs negatively regulate PI(3)P levels. Analysis of MTM expression patterns using transcriptional GFP reporters demonstrated that these two MTMs exhibit mostly nonoverlapping expression patterns and that MTM-GFP fusion proteins are localized to different subcellular locations. These observations suggest that some of the different functions of MTMs might, in part, be a consequence of unique expression and localization patterns. However, our finding that at least three CeMTMs play essential roles in coelomocyte endocytosis, a process that also requires vps34, indicates that MTMs do not simply turn off vps34 and unexpectedly also function as positive regulators of biological processes.