Analysis of a buried deposit in the Diamond Valley of southern California has revealed well-preserved pollen, wood, and diatom remains. Accelerator mass spectrometry dates of 41,200±2100 and 41,490±1380 14C yr B.P. place this deposit in marine isotope stage 3. Diatoms suggest a shallow lacustrine environment. Pollen data suggest that several plant communities were present near the site, with grassland, scrub, chaparral, forest, and riparian communities represented. Comparison with modern pollen suggests similarities with montane forests in the nearby San Bernardino and San Jacinto ranges, indicating vegetation lowering by at least 900 m elevation and temperatures 4°–5°C cooler than today. An increase in high-elevation conifer pollen documents climatic cooling near the profile top. Early-profile diatoms are typical of warm water with high alkalinity and conductivity, whereas later diatoms suggest a higher flow regime and input of cooler water into the system. We suggest that the sequence is part of the cooling phase of an interstadial Dansgaard–Oeschger cycle. Records of the middle Wisconsin period are rare in southern California, but the Diamond Valley site is similar to records from Tulare Lake in the San Joaquin Valley and the ODP Site 893A record from Santa Barbara Basin. It is probable that the Diamond Valley assemblage is a local expression of a vegetation type widespread in the ranges and basins of southwestern California during the middle Wisconsin.