The effect of the alkyl-chain substitution on ordering and phase behavior of monoglycerol esters is studied by coupling the results of surface pressure-area (pi-A) isotherm measurements, Brewster angle microscopy, and grazing incidence X-ray diffraction. In the present work, structure features and phase behavior of 1-(12-hydroxy)stearoyl-rac-glycerol monolayers are investigated and compared with those of the nonsubstituted I-stearoyl-rac-glycerol and 12-hydroxystearic acid monolayers. The dominating effect of the alkyl-chain substitution by a hydroxyl group in the 12 position on the monolayer properties is demonstrated. Phase behavior, domain morphology, and two-dimensional lattice structure of the two 12OH-substituted amphiphiles are similar to each other but different from those of the usual nonsubstituted amphiphiles. Main characteristics of the 1-(12-hydroxy)stearoyl-rac-glycerol monolayers are an extended flat phase-transition (plateau) region of the pi-A isotherms related with a small temperature dependence of the main phase-transition points, domains with several arms which reflect homogeneously and grow rather irregularly with different tendencies to form curvatures and oblique lattice structures. The bipolar character of amphiphiles OH substituted in the midposition of the alkyl chain is obviously responsible for the special monolayer characteristics deviating from those of typical amphiphilic monolayers.