Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO³₄, and Mg²⁺ were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K⁺ and total CO₂ (totCO₂). Na⁺ concentration ([Na⁺]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO₂ concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K⁺], but the interpretation of the results for [Na⁺] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.