N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-Q) is a derivative of rubber tires which leaches into surface waters when tire particles are swept into roadway runoff. 6PPD-Q has been identified as a potential cause of urban runoff mortality syndrome in coho salmon, and subsequent research has determined a wide variation in toxicity among fishes. While adult rainbow trout are known to be sensitive, there is limited research on the response of early life stages to 6PPD-Q exposure. Given that early life stages of fish are often more sensitive than adults, the aim of these studies was to assess the acute and subchronic toxicity of 6PPD-Q in early life stage rainbow trout. Rainbow trout alevins were exposed from hatch until 28 days post-hatch to time-weighted average 6PPD-Q concentrations ranging from 0.06 to 2.35 μg/L, which determined a 28-day median lethal dose (LC50) of 0.56 μg/L, as well as morphological deformities, including pooling of blood in the caudal fin. A follow-up acute study with exogenously feeding rainbow trout fry revealed a 96-h LC50 of 0.47 μg/L. These studies indicate that early life stage rainbow trout are sensitive to subchronic 6PPD-Q exposure, and underscore the importance of utilizing early life stage studies to determine the most sensitive benchmark concentrations, and their value in determining sublethal effects.