In order to study the preservation of phospholipids in specimens for electron microscopic study, two procedures were compared; routine double fixation with glutaraldehyde followed-by osmium tetroxide and fixation with a mixture of tannic acid-glutaraldehyde followed by osmium tetroxide, utilizing both glass slide smear and ultrathin section methods, using artificial lung surfactant (mainly composing of phospholipids). The following results were obtained. (1) In routine double fixation with glutaraldehyde-osmium tetroxide, although the lamellar structure, mainly composed of phospholipids, was often visualized, prefixation with mixed tannic acid-glutaraldehyde always resulted in a lamellar structure with a regular periodicity and good contrast. (2) The saturated phospholipid was better preserved with acetone dehydration than with alcohol dehydration. (3) Depending on the outcome of the first fixation, there was extensive loss of phospholipids during the process due to alcohol dehydration and propylene infiltration. (4) When the specimens were fixed with osmium tetroxide prior to tannic acid treatment, the multilamellar structure of the bilayer was usually irregular. Moreover, if the specimens were fixed with osmium tetroxide without tannic acid, phospholipid preservation was not good. From the above results, it became, apparent that prefixation by a mixture of tannic acid-glutaraldehyde followed by osmium tetroxide postfixation and dehydration by acetone was the most appropriate method for preserving saturated phospholipids and thus a stable ultrastructure of phospholipids an lamellar will obtained.