Plasma cholesterol esterifying activity has been measured in guinea pigs fed either a control diet or the same diet supplemented with 1% cholesterol. The extent of esterification was found to be similar in the cholesterol-fed and control guinea pigs and somewhat lower than in rats. The initial rate of esterification was also of the same magnitude as that found in rats and humans, and unaffected by dietary cholesterol if autologous plasma was used as substrate. However, LCAT activity from cholesterol-fed guinea pigs was significantly higher than that of control plasma when acting on either control or cholesterol-fed substrate. This suggests that dietary cholesterol increases the amount (or activity) of LCAT but that the substrate is unsuitable or that a necessary cofactor is present in limiting amounts. Heat treatment of guinea pig plasma seems to alter substrate availability to varying degrees. The implications of these findings in relation to substrate specificity and cofactor requirements of guinea pig LCAT are discussed.