We investigated the ability of strontium isotope ratios (⁸⁷Sr/⁸⁶Sr) in otolith cores to record the natal habitats of juvenile delta smelt Hypomesus transpacificus from the San Francisco Estuary, USA. Young delta smelt (<60 days old) were collected during the California Department of Fish and Game 20-mm Survey in May and June of 1999 at several potential natal areas: Napa River, Suisun Marsh, West Delta, North Delta, Central Delta, South Delta and East Delta. The core region of sagittal otoliths was assayed with laser ablation-multicollector inductively coupled plasma mass spectroscopy. The laser ablation technique provided precise estimates of ⁸⁷Sr : ⁸⁶Sr ratios with relative standard deviation of 0.003% (one sigma). Isotope ratios ranged from 0.7065 to 0.708 and were different among natal habitats. However, natal habitats within the delta region were not discernable among each other, and reflect the mixing of the two major rivers, Sacramento River and San Joaquin River within the delta. We will therefore be able to determine natal habitats for delta smelt by assaying the core region of the otoliths. The application of strontium isotope ratios (⁸⁷Sr/⁸⁶Sr) in fish otoliths will greatly improve conservation efforts for this protected species.