A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA⁺ MR cell, was found to accumulate over 3-fold more ¹⁰⁹Cd than did PNA^- MR cells, pavement cells (PV cells) and mucous cells, during a 1-hour in vivo exposure at 2.4 µg/L ¹⁰⁹Cd. In vitro ¹⁰⁹Cd exposures, performed in standard phosphate-buffered saline (PBS) and Cl^- -free PBS, at concentrations from 1 to 16 µg/L ¹⁰⁹Cd, were also carried out to further characterize Cd uptake kinetics. As was observed during in vivo experiments, PNA⁺ MR cells accumulated significantly more ¹⁰⁹Cd than did other cell types when exposures were performed using an in vitro procedure in PBS. Under such conditions, Cd accumulation kinetics in all cell types could be described using Michaelis-Menten relationships with Km values of approximately 3.0 µg/L Cd (27 nM) for both MR cell subtypes and 8.6 µg/L Cd (77 nM) for PV cells. In similar experiments performed in Cl^- free conditions, a significant reduction in ¹⁰⁹Cd accumulation in PNA⁺ MR cells was seen, but not in PNA^- MR nor PV cells. In vitro ⁴⁵Ca fluxes were also performed to determine the cellular localization of Ca transport in these functionally distinct populations of gill cells. ⁴⁵ Ca uptake was most pronounced in PNA⁺ MR cells, with levels over 3-fold higher than those found in either PNA^- MR or PV cells. Results from the present study suggest that the PNA⁺ mitochondria-rich cell type is a high-affinity and high-capacity site for apical entry of cadmium and calcium in the gill epithelium of rainbow trout.