A chemical, electrophysiological and behavioral study was carried out to analyze the volatile compounds of the Brindley’s gland of Rhodnius prolixus Stål. Six compounds were found in the Brindley’s gland of males and females: acetic, isobutyric, caproic acids and three compounds not identified. The mass spectra of the unidentified compounds have the appearance of a propionate ester, a butyrate ester and a valerate ester. There was no difference in the concentration of these compounds in the glands of males and females. The major component was isobutyric acid. The electroantennographic (EAG) evaluation of isobutyric, acetic, and caproic acids elicited weak responses. With both sexes, the EAG responses for the acids were no different from the control response at any of the doses evaluated (1, 10, 100, and 500 μg). The behavioral response of males and females to acetic, isobutyric, and caproic acids at 1, 5, and 10 μg and binary and tertiary mixtures of the three compounds was evaluated in a Y-olfactometer. Males showed preference for acetic acid at a dose of 1 μg, but not at 5 or 10 μg. Females showed preference for this compound at the dose of 5 μg. Males showed preference for isobutyric acid at 1 and 5 μg, but not at 10 μg. Females did not show any preference for isobutyric acid. Males were attracted to caproic acid at the dose of 1 and 10 μg. Females were attracted to the caproic acid. Males, but not females, preferred the mixture of acetic and isobutyric acids over control. Neither males nor females showed preference for the mixture of acetic and caproic acids or hexane control. Females, but not males, were attracted to the mixture of the isobutyric and caproic acids. Only males showed a positive response for the tertiary mixture of the acids.