Lemna species are widely used in ecotoxicological research to assess the phytotoxicity of environmental compounds. The purpose of the work described in this paper was to further refine the Lemna spp. bioassay, making it more robust and useful in comparing results between laboratories. A nondestructive image analysis method was used to measure growth as affected by herbicides during a time course. We tested 26 herbicides, with as many as 19 different modes of action, on leaf area growth of Lemna paucicostata to establish complete dose-response relationships. By using 3,5-dichlorophenol as a reference compound, different EC50 (concentration that inhibited growth by 50%) values of the herbicides can be compared. The EC50 values ranged from 0.003 microM for sulcotrione to 407 microM for asulam. Complete dose-response parameters were obtained that will better allow comparison of these results to those of other laboratories. These results should give useful information to anyone who works with Lemna species to address questions on herbicide residues in environmental compartments, screening of new herbicidal compounds, or the assessment of phytotoxic side effects of any compound.