The chromophore of the retinal-protein bacteriorhodopsin undergoes Light-induced isomerization around the 13-14 double bond and senses protein conformational changes. These events are accompanied by changes in the orientation of the electronic transition dipole moment and may be conveniently monitored by time-resolved polarized absorption spectroscopy. We used samples of oriented immobilized purple membranes and excited them isotropically. This has a number of advantages over photoselection with suspensions of membranes : (1) a high signal to noise ratio, (2) absence of depolarization due to rotational diffusion, and (3) precise determination of the angle of the transition dipole moment and the membrane normal. We show that with isotropic excitation the anisotropy of a transient intermediate i is given by r(i) = S2P2(cos theta(i)), in which S-2 is the orientational order parameter of the membranes and theta(i) the angle of the transition dipole moment with respect to the membrane normal. S-2 may be obtained from the steady-state anisotropy of the initial state. This approach was applied to determine the transition dipole orientation in the O-intermediate. Purple membranes were oriented in a 14-T magnetic field and immobilized in a polyacrylamide gel. S-2 was 0.74. : (1) a high signal to noise ratio, (2) absence of depolarization due to rotational diffusion, and (3) precise determination of the angle of the transition dipole moment and the membrane normal. We show that with isotropic excitation the anisotropy of a transient intermediate i is given by r(i) = S2P2(cos theta(i)), in which S-2 is the orientational order parameter of the membranes and theta(i) the angle of the transition dipole moment with respect to the membrane normal. S-2 may be obtained from the steady-state anisotropy of the initial state. This approach was applied to determine the transition dipole orientation in the O-intermediate. Purple membranes were oriented in a 14-T magnetic field and immobilized in a polyacrylamide gel. S-2 was 0.74.