Uncertainty and variability affect economic and environmental performance in the production of biotechnology and pharmaceutical products. However, commercial process simulation software typically provides analysis that assumes deterministic rather than stochastic process parameters and thus is not capable of dealing with the complexities created by variance that arise in the decision-making process. Using the production of penicillin V as a case study, this article shows how uncertainty can be quantified and evaluated. The first step is construction of a process model, as well as analysis of its cost structure and environmental impact. The second step is identification of uncertain variables and determination of their probability distributions based on available process and literature data. Finally, Monte Carlo simulations are run to see how these uncertainties propagate through the model and affect key economic and environmental outcomes, Thus, the overall variation of these objective functions are quantified, the technical, supply chain, and market parameters that contribute most to the existing variance are identified and the differences between economic and ecological evaluation are analyzed. In our case study analysis, we show that final penicillin and biomass concentrations in the fermenter have the highest contribution to variance for both unit production cost and environmental impact. The penicillin selling price dominates return on investment variance as well as the variance for other revenue-dependent parameters. (c) 2004 Wiley Periodicals, Inc.