Fluorescence correlation spectroscopy (FCS) was used to determine diffusion coefficients (D) of the Suwannee River fulvic and humic acids (SRFA and SRHA) and to measure the effects of pH, ionic strength, Ca, and humic substance (HS) concentration on the value of D. For these HS, average diffusion coefficients were in the range of (2−3) × 10-10 m2 s-1 (corresponding to hydrodynamic diameters of approximately 1.5−2.1 nm). Small, but significant, decreases in the diffusion coefficients were observed with decreasing pH, most likely indicating that a small degree of aggregation (formation of dimers and trimers) was occurring. The effect of ionic strength (up to 100 mM) was either small or insignificant, but, where a change occurred, there was a tendency toward a reduction in the diffusion coefficient with increasing salt concentration. No effect of HS concentration (1−50 mg L-1) or hydration time (1−14 days) was observed. There were no observable effects of Ca in comparison to Na, at an ionic strength of 5 mM. For all conditions studied, SRFA had a higher diffusion coefficient than SRHA which is in agreement with literature data on their respective molar masses. Since this is the first application of FCS to HS, the technique was systematically tested for artifacts, in particular photo bleaching and the effect of fluorescence excitation wavelength. Despite a small amount of photobleaching due to the intense laser excitation, FCS was found to be suitable for use with fulvic or humic acids