A multiyear research effort at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) evaluated several commercially available nitrate reductase (NaR) enzymes as replacements for toxic cadmium in longstanding automated colorimetric air-segmented continuous-flow analyzer (CFA) methods for determining nitrate plus nitrite (NOx) in water. This research culminated in USGS approved standard- and low-level enzymatic reduction, colorimetric automated discrete analyzer NOx methods that have been in routine operation at the NWQL since October 2011. The enzyme used in these methods (AtNaR2) is a product of recombinant expression of NaR from Arabidopsis thaliana (L.) Heynh. (mouseear cress) in the yeast Pichia pastoris. Because the scope of the validation report for these new automated discrete analyzer methods, published as U.S. Geological Survey Techniques and Methods 5-B8, was limited to performance benchmarks and operational details, extensive foundational research with different enzymes-primarily YNaR1, a product of recombinant expression of NaR from Pichia angusta in the yeast Pichia pastoris-remained unpublished until now. This report documents research and development at the NWQL that was foundational to development and validation of the discrete analyzer methods. It includes: (1) details of instrumentation used to acquire kinetics data for several NaR enzymes in the presence and absence of known or suspected inhibitors in relation to reaction temperature and reaction pH; and (2) validation results-method detection limits, precision and bias estimates, spike recoveries, and interference studies-for standard- and low-level automated colorimetric CFA-YNaR1 reduction NOx methods in relation to corresponding USGS approved CFA cadmium-reduction (CdR) NOx methods. The cornerstone of this validation is paired sample statistical and graphical analysis of NOx concentrations from more than 3,800 geographically and seasonally diverse surface-water and groundwater samples that were analyzed in parallel by CFA-CdR and CFA enzyme-reduction methods. Finally, (3) demonstration of a semiautomated batch procedure in which 2-milliliter analyzer cups or disposable spectrophotometer cuvettes serve as reaction vessels for enzymatic reduction of nitrate to nitrite prior to analytical determinations. After the reduction step, analyzer cups are loaded onto CFA, flow injection, or discrete analyzers for simple, rapid, automatic nitrite determinations. In the case of manual determinations, analysts dispense colorimetric reagents into cuvettes containing post-reduction samples, allow time for color to develop, insert cuvettes individually into a spectrophotometer, and record percent transmittance or absorbance in relation to a reagent blank. Data presented here demonstrate equivalent analytical performance of enzymatic reduction NOx methods in these various formats to that of benchmark CFA-CdR NOx methods.