Although two-component signaling systems, comprising a sensory histidine kinase and a response regulator, are a primary means by which bacteria detect and respond to environmental stimuli, they are poorly characterized. Here we report optimized conditions for detecting histidine phosphorylation using a facile medium-throughput filter paper-binding assay. Employing this assay we report the kinetic parameters of previously uncharacterized histidine kinases from Vibrio haveyi, Vibrio parahaemolytius, Shewanella oneidensis, and Legionella pneumophila. In characterizing these kinases, we effectively double the number of kinetically characterized histidine kinases that have been reported in the literature. (C) 2015 Elsevier Inc. All rights reserved.