Suramin has long been used in the treatment of various human diseases. Intravenous infusions of Suramin are commonly administered to patients over extended periods of time but there are a number of significant contraindications with peripheral muscle weakness being one of the most frequently reported. Previous work has shown that even after a single infusion (300 mg kg-1) Suramin remains in skeletal muscle in effective concentrations (11.6 μg mL-1; 84 days) for prolonged periods. These observations provide a strong rationale for investigation of the specific effects of Suramin on skeletal muscle function. Single mechanically skinned fibers were directly exposed to Suramin (10, 100 or 500 μmol L-1) for defined durations (2-10 min) in controlled physiological solutions that mimic the intracellular ionic environment of a fiber. Suramin treatment (10-500 μmol L-1) directly affected the contractile apparatus in a dose-dependent manner causing a decrease in Ca2+-sensitivity (pCa50 = -log (Ca2+) concentration, where 50% of maximum Ca2+- activated force is produced) by 0.14 to 0.42 pCa units and reduction in maximum Ca2+-activated force by 14 to 62%. Suramin treatment (100 μmol L-1 for 10 min and 500 μmol L-1 for 2 min) also caused development of a Ca2+-independent force corresponding to 2.89 ± 4.33 and 16.77 ± 7.50% of pretreatment maximum Ca2+-activated force, respectively. Suramin treatment (100 μmol L-1, 2 min) also increased the rate of sarcoplasmic reticulum (SR) Ca2+ release without significant changes in SR Ca2+ uptake. We report new functional effects for Suramin related to alterations in both the contractile apparatus and SR Ca2+-handling of skeletal muscle that may contribute to the peripheral muscle weakness noted in human pharmacological treatments.