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The objectives of the present study were to determine if the viability of freshwater mussel larvae (glochidia) is an ecologically relevant endpoint for toxicity tests and to define the appropriate duration of those tests. The authors assessed 1) how viability (the shell closure response to sodium chloride) compares with infectivity (ability to attach to a host fish and successfully metamorphose to the juvenile stage), and 2) the decline of viability and infectivity over time after glochidia were released from female mussels. Glochidia of 7 mussel species were isolated from females, placed in water, and subsampled daily for 2 d to 5 d. Viability, when 90%, was generally a good predictor of infectivity; however, when viability was <90%, infectivity was often disproportionately low, especially for glochidia collected near the end of the brooding period. Viability and infectivity declined more rapidly in natural water and sediment compared to reconstituted water. Following 24-h exposure to a toxicant (sodium chloride or copper), infectivity of the viable glochidia did not differ among concentrations of toxicants. The results indicate that viability is a valid proxy for infectivity and an ecologically relevant endpoint for standard toxicity tests with freshwater mussels for any test duration with control viability >90%. Environ Toxicol Chem 2014;33:199-207. (c) 2013 SETAC