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We examined the special characteristics of various kinds of fixatives and tried to find the most suitable method for simultaneous histological estimation of the mucous gel layer and mucous cells in rat gastric paraffin sections stained with Alcian blue (pH2.5)-periodic acid Schiff (AB-PAS). The tested fixatives were (group in parentheses): absolute ethanol at -80 degrees C (A), absolute methanol at -80 degrees C (B), Carnoy's solution (C), formalin-ethanol (D) and formalin-Tyrode (E). The thickness of the mucous gel layer (ML) and the numbers of AB- and PAS-positive cells (AB cells, PAS cells) of both the fundic gland area (F. area) and pyloric gland area (P. area) were measured microscopically (x 200). ML in the F. area was found in the order of (A) &gt; (B) &gt; (C) &gt; (D) = (E). AB cells were in the order of (A) &gt; (E) &gt; (B) &gt; (C) &gt; (D); and PAS cells were in the order of (B) &gt; (A) &gt; (E) &gt; (C) &gt; (D). Effects of various fixatives in the P. area showed the same trend as the F. area. Apart from above mentioned procedures, we observed the effects of ethanol at various temperatures: -80 degrees C, -25 degrees C, -4 degrees C and 20 degrees C. ML, AB and PAS cells showed the highest level at -80 degrees C. Moreover, to confirm that these fixatives retain the mucus, we evaluated the hexose and hexosamine contents contained in the fixative solution after fixation. As a result of various fixations, the hexose and hexosamine values in the fixative solution were the smallest for ethanol at -80 degrees C. In conclusion, ethanol at -80 degrees C was the most suitable fixative for histological estimation of mucous gel and mucous cells in rat gastric surface mucosa.