The overall goal of this research was to systematically evaluate H(2) production among different heterocystous cyanobacteria in response to defined experimental variables including N(2) and O(2) concentration, carbon source, and light intensity. N(2) elicited an immediate reduction of H(2) production rates and the magnitude of the effect was strikingly similar across the diverse collection of heterocystous cyanobacteria that were tested. At the N(2):O(2) ratio found in air (4:1), N(2) was a much more potent inhibitor of H(2) production than O(2). Low levels of O(2) (1-5% headspace, vol:vol) were generally found to support optimal H(2) production. Glucose addition (10 mM) stimulated light-dependent H(2) production in 8 of 10 cyanobacteria examined, eliciting a 2-11 fold increase in production rates and 2-45 fold increase in yields. The addition of glucose also effectively lowered the intensity of light required for optimal H2 production in 4 of 10 strains tested. H(2) production rates ranged from 1 to 50 mu mol mg chi a(-1) h(-1). The results from this study provide important benchmark phenotypes against which to evaluate newly discovered H(2)-producing heterocystous cyanobacteria, and we discuss how these findings highlight the necessity of a multi-parameter approach to comprehensively screen for superior H(2)-producing heterocystous cyanobacteria. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.