The purpose of this study was to determine the effects of daily and/or initial additions of varying Fe(II) and Fe(III) amounts, in combination with or without EDTA, on Emiliania huxleyi cultures. Pertaining to this specific aim, the growth rate (mu), cell abundance (X10(4) cells L-1), total chi a concentration ([tg L-1), chi a quota (pg cell-'), chi a:C ratio, Fe content (amol cell(-1)), Fe:C and Fe:chl a (pmol:ng) ratios, as well as pH values were observed. During the experiment, iron-free seawater was obtained by using a Chelex-100 column for iron-cleaning. The results revealed that the cell densities, growth rates, chlorophyll a concentrations, Fe contents, the calculated Fe:C ratios, and pH values were higher in the cultures containing 5000 nM Fe(III) EDTA than those of 5 nM Fe(III) and Fe(II). The growth rate, cell abundance, and total chi a concentration, on the other hand, were higher with daily additions of 5 nM Fe(II) instead of 5 nM Fe(III). Furthermore, cellular Fe quota was higher in the cultures with Fe(III). Cell abundance, chlorophyll a concentration and Fe:Chl a ratio were similar in 5 nMFe(III)+ EDTA cultures (initially or daily added), possibly be due to the negative effects of EDTA on both. The growth rate, cell abundance, total chi a concentration, and Fe quota were higher in cultures with inorganic Fe(II) and Fe(IH) contents, compared to those treated with EDTA containing Fe(II) and Fe(III). E. huxleyi cells seemed not to have used EDTA-bound iron, but their own released ligand to bind iron. Unexpectedly, while cellular iron content of cultures daily supplemented with Fe(II) was low, they exhibited the highest chi a content. The cell-chl a level was higher in daily supplemented cultures, with regard to those supported only initially with iron, evidencing the importance of continuous iron supply in chi a synthesis.