Fish hatcheries and other types of aquatic facilities are potential sources for the introduction of nonnative species of fish or aquatic invertebrates into watersheds. Chlorine has been suggested for use to kill organisms that might be released from the effluent of a facility. While acute LC50s (concentrations lethal to 50% of organisms exposed for up to 96 h) for chlorine are available for some species, short-term LC100s for chlorine have not been determined. The objective of this study is to establish concentrations of chlorine that are lethal to 100% of organisms after brief (1-, 5-, or 15-min) exposures. A total of 22 species were exposed to total residual chlorine concentrations (TRC) of 1, 10, or 25 mg TRC/L for 1, 5, or 15 min under static conditions followed by a 24-h postexposure recovery period in water without the addition of chlorine. Concentrations of chlorine resulting in 100% lethality of organisms were established for all of the species tested except for four species of mollusks or for a beetle. Exposures for 5 to 15 min to 10–25 mg TRC/L were the lowest combined time–chlorine treatments under which all of the fish tested and the other invertebrates tested (17 species) exhibited 100% lethality by the end of the initial chlorine exposures or after the 24-h recovery period.