Laboratory-based algal assays were developed to explore the bioavailability of copper to the marine alga Thalassiosira weissflogii. A calibration strategy was developed that avoided use of the synthetic ligand ethylenediaminetetraacetic acid (EDTA) in the Aquil growth medium, thereby allowing ambient metal speciation. In a comparison of T. weissflogii cells grown in Aquil medium with EDTA to medium containing no added copper, zinc, and less than 0.003 nM of EDTA, no significant growth differences were observed after 8 d, indicating adequate stored nutrients. A 30-h assay was selected as the optimal time frame after examination of data from concentration-response experiments. Using 65Cu stable isotope additions, parameters examined included growth, chlorophyll a, copper uptake, phytochelatin production, and dissolved organic carbon excretion. The T. weissflogii specific growth rates decreased from 1.36 d(-1)( at pCu (i.e., the negative logarithmic concentration of free Cu) = 8.8 to 0.56 d(-1) at pCu = 7.8, whereas intercellular copper concentrations increased from 13.6 to 70.1 fg/cell, respectively. Calculated values of the copper concentration that caused a 50% reduction in algal growth of pCu = 7.7 and copper per algal mass of 625 microg/g were established. Using an algal assay based on EDTA-free culture medium, along with trace-metal clean techniques, the effect of copper on T. weissflogii and the speciation of copper in marine waters can be studied.