Lyotropic phase transition behaviour of solution-grown mono-domain thin layered crystals of L- and DL-dipalmitoylglycerophosphocholines (dipalmitoyl-GPC) was investigated in a low hydration region at room temperature. X-Ray diffraction measurements and observations by a polarising microscope were performed on the same crystal. In the layer plane of the as-grown thin layered crystal of l- or dl-dipalmitoyl-GPC, which was considered to be dihydrated (phase II), an optical anisotropy was observed. Through a slight absorption of water, the dihydrated crystal showed a monotropic phase transition to a lyotropic mesophase (phase III), in which the optical axis was rotated by about 90° in the plane of the layer. Associated with this phase transition, anisotropic changes both in the unit cell and the crystal dimension occurred. Further absorption of water induced a second lyotropic phase transition for each of the two substances, where the optical anisotropy in the layer plane vanished (phase IV). Combined X-ray measurements suggested that the optical anisotropies observed in phase II and III were caused not only by the inclination of the long aliphatic chains but also by the arrangement of the polar groups of the molecules, and that the binding of water to dipalmitoyl-GPC slightly differed in the l- and dl-molecules in the thin layered crystals and also in as-received powder (phase 1) from as-grown crystals.