There is a clear relationship between the pelvic floor muscles and urinary systems, which relates to urgency, frequency, incontinence, pelvic pain, and bowel complaints. The specific mechanisms which relate these two systems are not clear. Improved understanding of the relation between the pelvic floor muscles and bladder function is clinically relevant in establishing effective treatments to such problems as incontinence, secondary to birth. The following tissues were collected from normal adult female rabbits: pubococcygeus (Pc) and ischiocavernosus/bulbospongiosus (Ic/Bs) pelvic floor muscles. Bladder body muscle and mucosa, bladder base muscle and mucosa, and leg skeletal muscle were also collected. The following enzymatic assays were performed on each tissue: citrate synthase (CS), sarcoplasmic-endoplasmic reticular ATPase (SERCA), and choline acetyltransferase (ChAT). CS and SERCA activities were significantly higher in the Pc compared with the Ic/Bs pelvic floor muscles, whereas the ChAT activity of the Ic/Bs was higher than that of the Pc muscle. Based on our results, the Pc muscle is expected to have a significantly greater capacity to contract and a higher metabolic activity than those of the Ic/Bs muscles. We believe that an understanding of the biochemical activities of these three biomarker enzymes in normal pelvic floor muscles is essential in evaluating the effects of specific experimental dysfunctions created in pelvic floor muscle activity.