Cyclocreatine, an analogue of creatine, inhibits tumor cell proliferation in vitro and in vivo. The effects of cyclocreatine in large C6 glioma multicellular spheroids were mapped here by magnetic resonance microscopy. Diffusion-weighted images of C6 glioma spheroids resolved the bright viable rim and the dark necrotic center. Sequential sets of diffusion images, following cyclocreatine administration, showed increasing self-diffusion coefficients of the intracellular water in the viable rim (0.49 x 10(-5) cm2/s for untreated spheroids, 0.62 x 10(-5) cm2/s after 48 h perfusion with 20 mM cyclocreatine). This fact correlated with cellular swelling apparent in histological sections. The radial distribution of cyclocreatine and soluble lipids across perfused C6 spheroids was measured by one-dimensional chemical shift imaging. Cyclocreatine accumulation was prominent throughout the viable cell layer, with no cyclocreatine accumulation in the necrotic center. In both cyclocreatine-treated and control spheroids the lipid signal was highest in the necrotic center and lower in the inner viable cell layer.