Two days after castration, urinary free felinine plus N-acetylfelinine decreased 24% in male cats, but, by day 5, the concentration had not decreased to that routinely found in males that have been castrated for several months. In a second experiment, three groups of castrated adult male cats received different subcutaneous injections: control (carrier), testosterone, testosterone plus estradiol. A fourth group of intact adult female cats received a testosterone injection. Urine was collected and analysed for free felinine, N-acetylfelinine and 3-methylbutanolglutathione. Baseline blood testosterone and estradiol concentrations were low during the pre-period, but increased sharply after hormone injections. The concentration of all three urinary metabolites increased as a result of testosterone injections with estradiol not modulating the effect. The effect of testosterone was not gender dependent. The concentration of free felinine, N-acetylfelinine and 3-methylbutanolglutathione in the urine remained low in the placebo control group throughout the study. The relative molar contribution of free felinine to the total amount of felinine containing compounds increased due to testosterone treatment, while the contribution of 3-methylbutanolglutathione and N-acetylfelinine decreased. Testosterone increases free felinine, N-acetylfelinine and 3-methylbutanolglutathione excretion in castrated adult male and intact female cats, whereas estradiol does not modulate this effect.