The initiation of further development is fundamental to the infectious processes of parasitic nematodes. We have examined early developmental activation of Trichinella spiralis larvae during host invasion, with particular emphasis on the timing of events. Using a novel approach, we have observed changes in tissue-specific transcriptional activity in live larvae during the infectious process with the fluorescent nucleic acid dyes SYTO12 and acridine orange. Simultaneously, the metabolic switch from anaerobic metabolism, characteristic of the infective stage, to aerobic metabolism, as found in the enteral stages, was tracked by measuring activities of the key regulatory enzymes phosphoenolpyruvate carboxykinase and pyruvate kinase, as well as isocitrate dehydrogenase (NADP) activity, and used as a co-indicator for developmental activation. Both metabolic enzyme activities and transcription patterns were found to change in response to host death, liberation from the nurse cell, and exposure to components of the host stomach environment. The results give a clear indication that the activation processes of T. spiralis infective larvae occur at a much earlier time than previously thought, and are stimulated upon liberation of the larvae from the nurse cell inside the host stomach.