N-cis-Parinaroyl ganglioside GM1 and N-trans-parinaroyl ganglioside GM1 were synthesized and characterized by HPLC, TLC, component analysis, absorbance spectroscopy, and proton NMR spectroscopy. Steady-state fluorescence anisotropy of the purified compounds, incorporated into phosphatidylcholine liposomes, was measured in the presence and absence of choleragen (cholera toxin) and choleragenoid (cholera toxin B subunit). In gel-phase liposomes, anisotropy measurements indicated that the motion of the parinaroyl ganglioside was not affected by addition of choleragen or choleragenoid. In fluid-phase liposomes, however, addition of toxin resulted in increased anisotropy (decreased rotational motion) of the fluorescent gangliosides. This decreased motion was not observed with other parinaroyl lipid probes, such as phosphatidylcholine, glucosylceramide, or free fatty acids, indicating that the effect was due to specific ganglioside/toxin interactions. Varying the amount of ganglioside or the amount of toxin suggested that the effect of toxin on probe motion was saturable at approximately 1 choleragen (or choleragenoid) molecule/5 ganglioside molecules. These results are consistent with previous hypotheses regarding the ganglioside/choleragen interaction and indicate that parinaroyl ganglioside probes will be useful in elucidation of the molecular details of this interaction.