The behavior of the surface-active material Gramicidin A at the air-water interface and in various solvents was examined. 1. 1. Absorbance at 283 nm as a function of concentration shows that Beer's law is obeyed in the range of 10 −6 to 5·10 −5 M gramicidin in ethanol. Peak fluorescence intensity is linear with gramicidin concentration in ethanol from 10 −8 to 10 −6 M. 2. 2. In heptane-water partition experiments, only a very small amount of gramicidin could be detected in either phase but as much as 80 % of the initial gramicidin could be recovered on the experimental vessel's walls. In this regard, the solubility of gramicidin in hexane was found to be less than 1·10 −6 M. Thus, gramicidin does not partition as favorably in media of low dielectric constant as do the presumed shuttle carriers, valinomycin and monactin. Solubility in aqueous media is also limited and leads to the growth of aggregates as indicated by increases in light scattering of gramicidin in water as compared to ethanol and by visible turbidity at concentrations above 5·10 −7 M. 3. 3. Gramicidin will form monolayers at the air-water interface. A force-area curve of gramicidin shows an initial rise at 350 Å 2, a gentle plateau at 12–16 dynes/cm and 150–180 Å 2 with an imprecise collapse at 18–23 dynes/cm and 130±10 A ̊ 2 . The latter molecular area is in agreement with the cross-sectional area of the helix proposed by D. W. U rry ( Proc. Natl. Acad. Sci. U.S., 68 (1971) 672) if the helix is assumed to be perpendicular to the interface. Gramicidin monolayers do not exhibit an elevated surface potential as does valinomycin in the presence of a 1 M KCl subphase. 4. 4. The rate of penetration by gramicidin of a lecithin monolayer and the adsorption of gramicidin at the air-water interface approach proportionality with subphase concentration at concentrations below 1·10 −7 M. The rate of adsorption at the air-water interface falls off strikingly with time, while penetration into lecithin monolayers is initially smaller but constant in time. The first-order lecithin monolayer penetration kinetics are consistent with the proposed intramembrane dimerization of gramicidin to form ion-conducting channels. 5. 5. In conclusion, the monolayer data are consistent with the channel mechanism proposed by Urry, and the partition experiments are in contrast to the partition properties of the presumed shuttle carriers.