The onset of insulin-dependent diabetes mellitus (IDDM) is often associated with the infiltration of pancreatic cells by macrophages. Upon activation, macrophages release nitric oxide (NO) and superoxide (O2). These species or their reactive intermediates can be cytoxic, mutagenic, or carcinogenic. Previous studies have reported both positive and negative effects of extracellularly generated NO on insulin secretion and viability of pancreatic cells. Inherent problems of several previous studies assessing the effects of NO on insulin secretion include unsteady state NO concentration exposures and the generation of other potentially damaging species. In this study, these problems were eliminated by using a modified experimental system in which NO delivery was achieved via diffusion across a gas-permeable tube and O2 delivery was maintained using an enzymatic reaction. The delivery rates were constant, leading to steady state concentrations of NO and O2 in the experimental system. Based on reaction kinetics, a model was developed to predict NO, O2, and peroxynitrite (ONOO-) concentrations during the experiment. This study showed that NO, O2, and ONOO- at predicted concentrations as high as 2.8 microM, 0.25 microM, and 0.1 nM, respectively, do not affect the insulin secretion rates of betaTC3 pancreatic cells over short times.