Aggregation of phenylacetylene macrocycles (PAMs) in solution has been studied by H-1 NMR spectroscopy and vapor pressure osmometry. The association constant for dimerization, K-assoc, has been determined by curve fitting the concentration dependence of H-1 NMR chemical shifts to a model for monomer-dimer equilibrium. The reliability of the NMR-determined aggregation constants and aggregate size have been independently verified by vapor pressure osmometry measurements. Thermodynamic parameters for association have been obtained from van’t Hoff analyses which show the aggregation to be favored enthalpically. The aggregation of PAMs bearing various endo- and exo-annular functional groups and PAMs of different geometry and ring size has been studied. The type of pendant functional groups and the manner in which these groups are arranged on the macrocycle is shown to strongly influence self-association. PAMs substituted with electron withdrawing groups (e.g., esters) are more strongly associated than those bearing electron donating groups (e.g., alkyl ethers) or macrocycles bearing a combination of the two substituents. The type of alkyl substituent on the ester or ether group is less important as long as the substituent is not branched and is exo-annular. Endo-annular alkyl ethers as well as branched exo-annular alkyl esters severely disrupt aggregation. Rigidity of the macrocycle also influences self-association. In contrast to hexameric macrocycles, similarly substituted open-chain oligomers and a nonplanar macrocycle show much weaker association. These findings are discussed in the context of face-to-face pi-pi interactions between aromatic rings. Consideration has also been given to pi-pi interactions between aromatic and ethynyl groups and between a pair of acetylenes, but these are concluded to be less significant based on an analysis of data from the Cambridge Structural Database.