In September 2011, we investigated the distribution and composition of dissolved and particulate phosphorus (P) pools throughout Lake Superior, a large P-limited freshwater ecosystem. Average seston particulate P (PP) concentrations in the deep chlorophyll maximum (DCM; 85 ± 28 nmol L-1) were significantly greater than in the epilimnion (63 ± 22 nmol L-1). In contrast, average particulate organic carbon (POC): PP (mol: mol) ratios showed the opposite pattern (DCM = 303: 1 vs. epilimnion = 455: 1). Mean seston nucleic acid-P concentrations were invariant between the epilimnetic (23 ± 18 nmol L-1) and DCM (26 ± 18 nmol L-1) layers, but significantly greater concentrations of intact polar membrane-derived phospholipids were found in the DCM (6 ± 2 nmol L-1) relative to the epilimnion (4 ± 2 nmol L-1). Phospholipids were a minor proportion of PP (7-14%) and total membrane lipids (< 30%). Rather, our results suggest that microbial flora of Lake Superior substituted non-phosphorus lipids for phospholipids. In the nitrogen (N)-rich waters, N-based betaine lipids dominated (39-42%) the lipid pool, and concentrations were significantly greater in the P-poor epilimnetic seston. Sulfolipids were also abundant and significantly greater in the epilimnion (7 ± 2 nmol L-1) than in the DCM (4 ± 2 nmol L-1), despite low sulfate concentrations relative to marine environments. Our results demonstrate for the first time the importance of plankton producing non-phosphorus lipids for phospholipids as a strategy for reducing cellular P inventories in lacustrine regimes.