The acetylene block technique was employed to study denitrification in intertidal estuarine sediments. Addition of nitrate to sediment slurries stimulated denitrification. During the dry season, sediment-slurry denitrification rates displayed Michaelis-Menten kinetics, and ambient NO(3) + NO(2) concentrations (</=26 muM) were below the apparent K(m) (50 muM) for nitrate. During the rainy season, when ambient NO(3) + NO(2) concentrations were higher (37 to 89 muM), an accurate estimate of the K(m) could not be obtained. Endogenous denitrification activity was confined to the upper 3 cm of the sediment column. However, the addition of nitrate to deeper sediments demonstrated immediate N(2)O production, and potential activity existed at all depths sampled (the deepest was 15 cm). Loss of N(2)O in the presence of C(2)H(2) was sometimes observed during these short-term sediment incubations. Experiments with sediment slurries and washed cell suspensions of a marine pseudomonad confirmed that this N(2)O loss was caused by incomplete blockage of N(2)O reductase by C(2)H(2) at low nitrate concentrations. Areal estimates of denitrification (in the absence of added nitrate) ranged from 0.8 to 1.2 mumol of N(2) m h (for undisturbed sediments) to 17 to 280 mumol of N(2) m h (for shaken sediment slurries).