Operator training simulators (OTS) are software tools for training process operators in large-scale industrial applications. Here, we describe the development, implementation and training of an OTS for a large-scale industrial plant for bioethanol production. The design of the OTS is based on conceptual analysis (previously reported by us in this journal) of various configuration alternatives and training procedures at the plant. In this article, we report on how the conceptual design is used in simulation models and graphical user interfaces and how the design is applied for training of operators in the real plant environment. The results imply that OTS would be time- and cost-efficient tools for application in the biotechnological industry.