Rhesus macaque is a widely used primate model organism. Its genome annotations are however still largely comparative computational predictions derived mainly from human genes, which precludes studies on the macaque-specific genes, gene isoforms or their regulations. Here we took advantage of histone H3 lysine 4 trimethylation (H3K4me3)'s ability to mark transcription start sites (TSSs) and the recently developed ChIP-Seq and RNA-Seq technology to survey the transcript structures. We generated 14013757 sequence tags by H3K4me3 ChIP-Seq and obtained 17322358 paired end reads for mRNA, and 10698419 short reads for sRNA from the macaque brain. By integrating these data with genomic sequence features and extending and improving a state-of-the-art TSS prediction algorithm, we ab initio predicted and verified 17933 of previously electronically annotated TSSs at 500-bp resolution. We also predicted approximately 10000 novel TSSs. These provide an important rich resource for close examination of the species-specific transcript structures and transcription regulations in the Rhesus macaque genome. Our approach exemplifies a relatively inexpensive way to generate a reasonably reliable TSS map for a large genome. It may serve as a guiding example for similar genome annotation efforts targeted at other model organisms. The Author(s) 2010. Published by Oxford University Press.2010This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.