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Objectives: To evaluate the effect ovariectomy (OVX) after 2 and 4 weeks on bladder function and biochemistry of the adult female rabbit urinary bladder. Methods: Twelve mature female rabbits were divided into 3 groups: control, 2-week ovariectomized, and 4-week ovariectomized. At the end of the experimental period, the following studies were performed: contractile studies on isolated strips; examinations of the activity of citrate synthase (a marker for mitochondrial function) and thapsigargin-sensitive calcium ATPase (a marker for sarcoplasmic reticular calcium uptake function); and quantification of Rho-kinase (ROK) α and β and myosin light chain kinase by Western blot analyses. Results: By 28 days after OVX, there were significant decreases in bladder weight, contractile responses, and citrate synthase and sarcoplasmic reticular calcium uptake activity. In addition, by 28 days following OVX the relative concentration of ROK α was significantly increased, whereas ROK β concentration was significantly decreased. Myosin light chain kinase was significantly reduced. Conclusions: Our study demonstrated that OVX contributed significantly to chronically decreased contractile function in the detrusor muscle of the female rabbit bladder, and this decrease, in turn, was mediated by decreased mitochondrial and sarcoplasmic reticulum function. These specific bladder dysfunctions could be related to the demonstrated decreased blood flow to the bladder muscle and mucosa and the increased generation of free radicals. Changes in smooth muscle regulatory proteins, especially myosin light chain kinase, may also play a role in contractile dysfunctions. [Copyright &y& Elsevier]