PURPOSE: To study the characteristics of diffusion magnetic resonance imaging (MRI) contrast in a rat brain BT4C glioma during progression of ganciclovir (GCV)-thymidine kinase gene therapy-induced programmed cell death (PCD) in vivo. MATERIALS AND METHODS: The trace of the diffusion tensor (Dav = 1/3TraceD), T2, and spin density were determined by MRI and the apparent diffusion coefficient (ADC) of water by diffusion nuclear MR (NMR) spectroscopy using largely varying b values and diffusion times (tD) at 4.7 T. Cell count and apoptotic cells were quantified by histological means. RESULTS: Decline in cell count was strongly associated with increase in both Dav and T2. Spin density ratio between tumor and contralateral parietal cortex increased with a very similar time course as Dav and T2, indicating net water gain into the eradicating tumor. Diffusion spectroscopy showed a nonmonoexponential signal decay at all tD values ranging from 14-192 msec. During PCD, the ADC of the component yielding fast diffusion coefficient (D1), as acquired with tD > or = 47 msec, increased with kinetics similar to those of Dav (tD = 4.8 msec). The fractional size of D1 increased by 10% to 15% throughout the entire tD range. Apparent water residence time of the slow diffusion component, D2, shortened from a value of 38.3 +/- 1.7 msec on day 0 to 33.4 +/- 0.5 msec by day 8. CONCLUSION: The present results show that reduced cell density and increased water content, leading to altered water microenvironment, are associated with increased water diffusion coefficient in eradicating gliomas as a result of PCD.