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The high cost of fish meal in tilapia diets warrants the potential use of cottonseed meal (CSM) as an alternative source of high quality protein. The effects of varying levels of CSM (0, 25, 50, 75 and 100%) as fish meal protein replacement on growth, blood parameters, spleen characteristics, free and bound gossypol in blood plasma, haemoglobin and haematocrit were determined in tilapia. Gossypol (C30H30O8) is a polyphenolic substance found in cottonseed that has known toxic effects in fish. Tilapias ( n = 219, average weight = 11.3 ± 3.9 g) were randomly distributed into 15, 32-L glass aquaria, representing five dietary treatments and three replicates per treatment. Each aquarium containing 13–16 fish was supplied with thermoregulated, recirculating water (27 ± 1 °C) at 1 L min−1 flow rate and photoperiod was constant (12 h L/12 h D). Fish fed 25–50% CSM protein replacement showed similar body weights and total lengths as the controls at the completion of the 16-week trial. Fish fed 75 and 100% CSM protein replacement showed a significant decline in body weight and total length. Fish fed 25–100% CSM protein replacement had significantly lower haematocrit and haemoglobin (ANOVA/LSD, P < 0.05) compared with levels in controls. The decline was most prominent in groups fed diets with 50–100% CSM protein replacement. Total and free gossypol concentrations of blood plasma significantly increased with increasing levels of CSM replacement ( P < 0.05). No gossypol was found in blood plasma of fish from the control group. The occurrence of immature and abnormal erythrocytes was significantly greater among fish fed 75 and 100% CSM diets compared with fish fed 0–50% CSM diets. Spleen-somatic index (spleen weight/body weight × 100) did not differ between control fish and fish fed 50–100% CSM diets. Spleen abnormalities, such as large depositions of haemosiderin and melanin pigments and proliferation of melano-macrophage centres, lymphocytic depletion of the white pulp areas (hypocellularity), and presence of vacuoles and necrotic areas were observed among fish fed 50–100% CSM protein diets. In general, the pathological effects of gossypol in tilapia (low haemoglobin and haematocrit levels, abundance of immature red blood cells or polychromatocytes, abnormal spleen morphology) were similar to the effects of vitamin E and/or vitamin C deficiencies observed in other studies. [ABSTRACT FROM AUTHOR]