Objective: To investigate cardiac phenotypes in mice deficient in the peptide hormone relaxin by gene targeting. Methods: Echocardiography and cardiac catheterization were performed on male and female relaxin deficient (Rlx^−/−) mice as well as heterozygous (Rlx^+/−) and wildtype (Rlx^+/+) littermates aged between 8 and 24 months. Collagen expression and content in the heart were analysed by real-time PCR, hydroxyproline assay and histology. Results: Heart rate, blood pressures, left ventricular (LV) dimensions, fractional shortening and maximal and minimal dP/dt did not differ significantly between the three genotypes of either gender at any age. However, 8–10-month-old Rlx^−/− males exhibited a greater transmitral flow velocity (A-wave) at the late LV diastolic phase. Male Rlx^−/− mice aged between 12 and 24 months had significantly higher LV end-diastolic pressures, a 30% increase in atrial weight and 10–30% increases in lung and liver weights. Male mice also showed an age-dependent increase (P<0.01) in LV collagen content that was more pronounced in Rlx^−/− than control littermates (P<0.01). Procollagen type-1 expression was also significantly higher in the LV of Rlx^−/− males compared with either Rlx^+/− or Rlx^+/+ males at 6, 9 and 12 months of age. Age-matched female Rlx^−/− mice did not display any of these cardiac phenotypes seen in Rlx^−/− males. Conclusions: Male Rlx^−/− mice had impeded LV diastolic filling and increased atrial weights, most likely due to an increase in ventricular collagen content and chamber stiffness. These phenotypes in the Rlx^−/− males were not observed in Rlx^−/− females, indicating the importance of other gender-related factors in cardiovascular function.