OBJECTIVES: To prospectively assess the effects of newly developed chronic conditions on changes in health-related quality of life (HRQoL) in postmenopausal women. DESIGN: Prospective cohort study. SETTING: Forty clinical centers in the United States. PARTICIPANTS: Women aged 50 to 79 enrolled in the Women's Health Initiative Observational Study during 1993-98 (N = 75,198). MEASUREMENTS: Onset of seven chronic conditions (hypertension, heart disease, stroke, cancer, osteoporosis, diabetes mellitus, arthritis), HRQoL based on the Medical Outcomes Study 36-item Short Form survey (SF-36), and change in HRQoL from baseline to Year 3 in scores on the Physical (PCS) and Mental (MCS) Component Summary of the SF-36. RESULTS: Each of the seven chronic conditions was statistically significantly associated with HRQoL for physical and mental health. Women with incident stroke had the greatest decline in HRQoL for physical and mental health (PCS: 10.2, 95% confidence interval (CI) = 8.3-12.1; MCS: 10.4, 95% CI = 8.8-12.1), which exceeded minimal clinically important differences (MCID). Having more new chronic conditions was associated with greater decrease in HRQoL. Declines in HRQoL in women with two or more chronic conditions exceeded MCID. Those with heart disease and stroke had the largest decline in physical health, and those with stroke and arthritis had the greatest decline in mental health. For all conditions other than stroke and diabetes mellitus, the more recently a condition was diagnosed, the greater the decrease in PCS. For stroke and cancer, a similar pattern was observed for MCS. CONCLUSION: The more conditions a woman developed, the greater reduction in HRQoL she experienced. Declines in HRQoL in women with stroke, cancer, and some combinations exceeded MCID. Findings from this study might lead to more-effective patient-centered approaches to health care for women with multiple chronic conditions.