Although steady-state anisotropy measurements of phase-sensitive probes provide a qualitative description of the phase behavior of biomembranes, there is little information about the physical state of lipid domains. We have developed a ground-state double hindered rotator model (DHR) for fluorescence anisotropy decay, in which probes possess separate rotational correlation times and r infinity in each phase. To validate the model, multifrequency differential phase angles (delta) and modulation amplitudes (lambda) were measured in a two-compartment cuvette with combinations of POPOP, TMA-DPH, and DPH in isotropic solvents and in DPPC liposomes. Rotational parameters obtained by fitting the DHR model were similar to those of a single hindered rotator model fitted to data obtained separately for each probe. As predicted by the model, negative delta and decreasing lambda with increasing modulation frequency were obtained when fluorophores in isotropic solvents were paired with fluorophores in DPPC liposomes. The rotational parameters of the phase-sensitive fluorophores cis-parinaric (cPnA) and trans-parinaric (tPnA) acid in DPPC/DMPC (1:0, 0:1, and 1:1) liposomes were determined at 15-40 degrees C. Two lifetimes (1 and 3 ns) were obtained above the phase transition temperature (Tc); greater than 95% of the fluorescence intensity was described by two lifetimes (3-9 and 12-32 ns) below Tc. Negative delta values were obtained when solid-phase lipid was present. r infinity varied from 0.26-0.32 below to 0.11-0.14 above Tc; at intermediate T, where two phases coexists, r infinity values were approximately 0.23 and approximately 0.31. These data indicate very hindered PnA rotation in solid-phase lipid.(ABSTRACT TRUNCATED AT 250 WORDS)