We studied the effect of the duration (0.5-48 h) of silver impregnation on the intensity of Bodian's silver staining using formalin-fixed, paraffin-embedded sections of a human brain. The silver ion (Ag+) and copper ion (Cu2+) in the silver protein solution were quantified simultaneously for treatments of various durations. Both the intensity of staining and the quantities of Ag+ and Cu2+ were greatly affected by the duration of silver impregnation. While the quantity of Ag+ considerably decreased during the first 4 h of impregnation, that of Cu2+ greatly increased. Only small changes were observed in both ions after 12 h. Neurofibrils or axons, and neurofibrillary tangles (NFTs) were clearly stained after 12-24 and 16-48 h of impregnation, respectively. Strong staining of these components was not observed for other durations of treatment. The amount of metallic copper in silver impregnation also affected both the intensity of staining and the quantities of Ag+ and Cu2+ in the silver protein solution. Ag+ and Cu2+ were also present in the gold trichloride acid solution in which the section was toned. These findings suggest that both Cu2+ derived from metallic copper and silver protein are deposited on sections during silver impregnation, that the amount of Cu2+ may determine the amount of silver protein deposited on the section, and that the reduced (metallic) form of silver and copper on the section may participate in gold toning. Thus, to achieve strong staining of a desired component, it is important to examine the conditions of silver impregnation (i.e. duration and amount of metallic copper). For strong staining of neurofibrils, axons and NFTs, optimal results are obtained by the addition of 5 g of metallic copper foil to 100 ml of 1% silver protein solution, and by 16-24 h of impregnation.