The upper Miocene to Pliocene Bouse Formation in the lower Colorado River trough, which consists largely of siltstone with basal tufa and marl, has been interpreted as estuarine on the basis of paleontology. This interpretation requires abrupt marine inundation that has been linked to early rifting in the Gulf of California and Salton trough. New strontium isotope measurements reported here from carbonates and invertebrate shells in the Bouse Formation reveal no evidence of marine water, but are consistent with deposition in a lake or chain of lakes fed by the Colorado River. Furthermore, the absence of a southward decrease in ⁸⁷Sr/⁸⁶Sr within the Bouse Formation does not support the estuarine model in which low ⁸⁷Sr/⁸⁶Sr marine Sr would have dominated the mouth of the hypothetical Bouse estuary. Elevation of originally marine ⁸⁷Sr/⁸⁶Sr in the Bouse Formation to its present level, due to postdepositional interaction with ground water, is unlikely because Sr from secondary calcite above, below, and within the Bouse Formation is consistently less radiogenic, not more, than Bouse marl and shells. In contrast to Bouse Sr, strontium from mollusks in tidal-flat and delta-front paleoenvironments in the contemporaneous Imperial Formation in the Salton trough and from the subsurface south of Yuma was derived from sea water and confirms the dominance of marine strontium near or at the mouth of the late Miocene to early Pliocene Colorado River. Inferred post–early Pliocene uplift of the Bouse Formation from below sea level to modern elevations of up to 550 m has been used to support a late Cenozoic uplift age for the nearby Colorado Plateau. This constraint on uplift timing is eliminated if the Bouse Formation is lacustrine.