Time-resolved fluorescence anisotropy of the single tryptophan (Trp) residue of melittin was measured to assess the orientational fluctuations of the protein in lipid membranes above and below the lipid phase transition temp. (Tt). In phospholipid vesicles contg. melittin, 3 relaxation times were obsd. at temps. above Tt, whereas only 2 relaxation times were obsd. below Tt; the slowest of the 3 relaxations in fluid membranes was absent in the ordered membranes. In planar multibilayer membranes at temps. above Tt, 2 relaxation processes were detected, corresponding to the fastest and slowest relaxations in the fluid vesicles, whereas at temps. below Tt only 1 relaxation was obsd. The fastest relaxation was attributed to internal motion of the Trp ring, the intermediate relaxation was attributed to internal motions of the melittin helix coupled to motions of the nonhelical C-terminal segment, and the slowest relaxation was attributed to the rigid-body motions of the Trp-contg. helical segment of melittin. Order parameters were also evaluated. The method described for evaluating these data may be of general interest for characterizing protein fluctuations in lipid membranes. [on SciFinder (R)]