Methylation of lysine 79 on histone H3 (H3K79) is mediated by a methyltransferase called Dot1-like protein (DOT1L). DOT1L is involved in the regulation of telomeric silencing, development, cell cycle checkpoint and transcription. However, the mechanisms by which DOT1L controls these unrelated and diverse functions are unknown. To gain greater insight into DOT1L-mediated functions, we have purified a DOT1L-containing complex using tandem affinity purification. Mass spectrometry of the DOT1L-containing complex revealed that AF9, ENL and NPM1 were shown to be major DOT1L-interacting proteins. To construct a plausible DOT1L-interaction web, AF9-, ENL- and NPM1-containing complexes were also purified for mass spectrometry analysis. The data showed that DOT1L might control AF9- and ENL-mediated transcription, regulate RNA processing, and function as a histone chaperone in a NPM1-dependent manner. In addition, the purification of protein complexes identified a number of novel interacting partners associated with DOT1L, AF9, ENL and NPM1. These data define a unique DOT1L network and shed light on unknown functions of the DOT1L complex.