cGαo-/-females are not attracted to familiar male urine in a two-choice ownership recognition test. (A) To evaluate attraction and ownership recognition, a two-step experiment is performed: in an identity-learning phase (step 1: exposure), females are exposed to filter papers with urine streaks from two B6 males, one allowing direct physical contact (male #1 urine) and a second male urine (male #2) deposited on a filter paper in a meshed plastic cassette to prevent direct physical contact. In the recognition phase (step 2: recognition), the same females are given a choice between male #1 and #2 urine volatiles (no direct contact). (B) Preference for the previously contacted urine displayed by B6 females is absent in cGαo-/- female mice in step 2 (t-test: ***P <0.001 (B6); non-significant (ns) P = 0.19 (cGαo-/-)). (C) Preference in control B6 females is induced only by high molecular weight (HMW, >10 kDa) fraction of male urine in step 2 (t-test: P = 0.17 (low molecular weight (LMW)), **P <0.01 (HMW)). HMW activity is lost when tested on cGαo-/- females (t-test: P = 0.59). (D) HMW fraction is incubated with menadione, a competitive displacer that releases hydrophobic small volatile molecules from the major urinary proteins β-barrel binding pocket. Menadione-incubated HMW (mHMW) fraction prevents the formation of a preference in step 2 in wild-type animals (t-test: P = 0.54). (E) Both B6 and cGαo-/- females are able to discriminate urine volatiles from two different B6 males in a habituation-dishabituation paradigm (analysis of variance (ANOVA): F1,97 = 2.77, P = 0.1; least significant difference (LSD): P <0.005). (F) Preference for male versus female urine volatiles in a two-choice preference test is maintained intact in cGαo-/- females (ANOVA: F1,31 < 0.001, P = 1 ; LSD: ***P <0.001). ns, non-significant.