The accurate reconstruction of gene regulatory networks from large scale molecular profile datasets represents one of the grand challenges of Systems Biology. The Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) represents one of the most effective tools to accomplish this goal. However, the initial Fixed Bandwidth (FB) implementation is both inefficient and unable to deal with sample sets providing largely uneven coverage of the probability density space. Here, we present a completely new implementation of the algorithm, based on an Adaptive Partitioning strategy (AP) for estimating the Mutual Information. The new AP implementation (ARACNe-AP) achieves a dramatic improvement in computational performance (200× on average) over the previous methodology, while preserving the Mutual Information estimator and the Network inference accuracy of the original algorithm. Given that the previous version of ARACNe is extremely demanding, the new version of the algorithm will allow even researchers with modest computational resources to build complex regulatory networks from hundreds of gene expression profiles.