In this paper we show that a highly conserved aromatic residue, phenylalanine at the 340-position, is essential for ergoline binding to 5-hydroxytryptamine2A receptors. We hypothesized that F340 was essential for a specific aromatic-aromatic interaction (e.g., pi-pi or hydrophobic) between the phenyl moiety of F340 and the aromatic ring of the ergoline nucleus. To test this hypothesis, eight point mutations of adjacent (F340 and F339) and nonadjacent (F125) phenylanaines were made, using conservative (phenylalanine to tyrosine) and nonconservative (phenylalanine to leucine, alanine, or serine) substitutions. The binding affinities of all of the tested simple ergolines were greatly reduced by specific mutations of F340 in which aromatic-aromatic interactions (e.g., F340A and F340L) were abolished, but they were unaffected when the replacement residue was aromatic (e.g., F340Y). In contrast, the binding affinities of four ergopeptines (bromocryptine, ergocryptine, ergocornine, and ergotamine) were relatively unaffected by the F340L substitution. Neither ergoline nor ergopeptine affinities were consistently altered by F339 mutations. These results support the notion that aromatic-aromatic interactions (either pi-pi of hydrophobic) with F340 are essential for the binding of simple ergolines but not ergopeptines to 5-hydroxytryptamine2A receptors. Our findings support models of ergoline and ergopeptine binding to serotonin receptors that suggest that the nature of the substituent at the 8-position of the ergoline nucleus may give rise to different modes of binding for the two classes of agents, particularly with respect to the phenyl ring of F340.