UNLABELLED: Several clinical observations have suggested that HMPAO cerebral uptake might be related not only to regional cerebral perfusion but also to the nature of the lesion. Our aim was to investigate at the cellular level the nature of the process(es) involved in HMPAO accumulation in vitro. METHODS: Time-course incorporation of HM-PAO was studied in a fast-growing human premonocytic line, U937, in a human astrocytic-derived cell line, U373 and a human hybridized endothelial cell line, EaHy926. Minimal differences of HMPAO retention between these cell lines were observed and plateau of %U(HMPAO) (cpm cells/cpm standard of injected) were achieved within 2 hr. Because HMPAO cell retention was related to the intracellular content in glutathione, experiments studying effects of redox were conducted by preexposing U937 cells to D, L dithiothreitol or 2-Mercaptoethanol. RESULTS: Overnight incubation with NAC or BSO did not significantly modified the kinetic of 99mTc-HMPAO incorporation while overnight incubation with NAC resulted in a 2-fold increase in intracellular glutathione content and overnight incubation with BSO nearly abolished the intracellular glutathione content. At the opposite, presence of these reducing agents in the medium during the experiments completely abolished 99mTc-HMPAO retention. CONCLUSION: Our data thus provide in vitro evidence to support that overall intracellular retention of HMPAO is more dependent upon the redox activity of the tissue than the intracellular glutathione content. SPECT-HMPAO may accurately reflect regional cerebral blood flow in a normal state but possibly not in all pathological situations in which cell metabolism disturbances are characterized by alterations in the redox status.