Transformed, insulin-secreting endocrine cells have been proposed as an alternative to islets for the development of a bioartificial pancreas. With appropriate immunoprotection, such cells may be implanted without the need for patient immunosuppression. Use of continuous cell lines alleviates the cell availability limitation, but poses questions regarding the stability and biochemical and secretory function of the preparation, especially in the longterm. We have developed a bioreactor/perfusion perfusion system, compatible with a horizontal-bore NMR instrument, that can maintain immunoprotected endocrine cells for prolonged periods of time. P-31 NMR spectroscopy was used to study the bioenergetics of recombinant, insulin-secreting mouse pituitary AtT-20 cells entrapped as spheroids in calcium alginate/poly-L-lysine/alginate beads. NMR provided data verifying the macroscopic homogeneity within the bioreactor and allowing the evaluation of changes in cellular bioenergetics for a period of 70 days under different culture conditions. Levels of high-energy phosphates changed slightly during the first 40 days of the experiment, then decreased considerably as cell death occurred. Rates of glucose consumption and insulin-related peptide secretion also remained constant for 40 days and decreased rapidly thereafter. This study constitutes the beginning of an extensive quantitative analysis of the biochemistry of transformed endocrine cell lines in a sequestered, artificial tissue environment.