We produced eight anti-huntingtin (Htt) monoclonal antibodies (mAbs), several of which have novel binding patterns. Peptide array epitope mapping shows that mAbs MW1-6 specifically bind the polyQ domain of Htt exon 1. On Western blots of extracts from mutant Htt knock-in mouse brain and Huntington's disease lymphoblastoma cell lines, MW1-5 all strongly prefer to bind to the expanded polyQ repeat form of Htt, displaying no detectable binding to normal Htt. These results suggest that the polyQ domain can assume different conformations that are distinguishable by mAbs. This idea is supported by immunohistochemistry with wild type (WT) and mutant Htt transgenic mouse (R6) brains. Despite sharing the same epitope and binding preferences on Western blots, MW1-5 display distinct staining patterns. MW1 shows punctate cytoplasmic and neuropil staining, while MW2-5 strongly stain the neuronal Golgi complex. MW6, in contrast, stains neuronal somas and neuropil. In addition, despite their preference for mutant Htt on blots, none of these mAbs show enhanced staining of R6 brains over WT, and show no binding of the Htt-containing nuclear inclusions in R6 brains. This suggests that in its various subcellular locations, the polyQ domain of Htt either takes on different conformations and/or is differentially occluded by Htt binding proteins. In contrast to MW1-6, MW7, and 8 can differentiate transgenic from WT mice by staining nuclear inclusions in R6/2 brain; MW8 displays no detectable staining in WT brain and stains only inclusions in R6/2 brain. Epitope mapping reveals that MW7 and 8 specifically bind the polyP domains and amino acids 83-90, respectively. As with MW1-6, the epitopes for MW7 and 8 are differentially available in the various subcellular compartments where Htt is found.