The spatiotemporal chemical characteristics of the water column in Linsley Pond, a freshwater lake in Connecticut, were studied from just before its stratification in April through the fall turnover in December. Two low-molecular-weight thiols, glutathione and 3-mercaptopropionic acid (MPA), were detected at nanomolar concentrations in the water column. GSH was detected (average concentrations were ca. 5 nmol L21 in the particulate phase and ca. 3 nmol L21 in the dissolved phase) in surface and near-surface waters only and covaried with chlorophyll a. Both particulate and dissolved MPA were measured in the oxic and anoxic regions. In the metalimnion, MPA concentrations were greater than in the oxic water layers above. At times, the MPA concentrations in the metalimnion were as high as those found in the anoxic hypolimnion. The MPA present at different depths in the Linsley Pond water column is most likely produced through dissimilar mechanisms. Throughout the water column, MPA may be a product of the metabolic degradation of sulfur-containing organic compounds; however, in the hypolimnetic waters, dissolved sulfide may play an important role in MPA formation through abiotic nucleophillic addition to unsaturated functionalities in dissolved organic matter. Parallel laboratory experiments were performed to assess the importance of MPA in copper speciation. The results confirmed that despite nanomolar levels in Linsley Pond, MPA does not play an important role in copper speciation. A survey of six additional lakes in Connecticut was made for detectable thiols. Only MPA was detected in four of these lakes, and its distribution was similar to that in Linsley Pond.