AbstractSymbiosis with micro‐algae (photosymbiosis) is a common feature among marine planktonic protists, but very little is known about the physiology and ecological significance of these associations. High concentrations of dimethylsulfoniopropionate (DMSP), a metabolite produced by marine microalgae, are commonly observed in coral‐microalgae symbiosis, where DMS(P) is involved in multiple physiological functions. Knowledge on concentration and role of DMSP in analogous photosymbiosis in plankton is lacking. Here, we assess the total DMSP (DMSPt = DMSP + DMS) concentration and natural stable isotopes of sulfur across ecologically relevant symbiotic plankton groups, the Radiolaria and Foraminifera. We found that intracellular DMSPt concentrations in microalgal symbionts were among the highest recorded (range = 170–702 mmol L−1), while lower concentrations (range = 0.1–23 mmol L−1) were characteristic of the holobiont (i.e., host‐microalgae). The contribution of symbiotic Radiolaria to the water column particulate DMSPt concentration ranged 0.1–8%. Sulfur isotopic composition (34S) of DMSPt in the Collodaria holobionts was significantly higher than their symbiotic microalgae isolated in culture. Despite their high intracellular DMSPt content, uptake in these holobionts throughout 3‐d incubations was not detected. We observed a systematic 34S depletion (∼ 1.5‰) of DMS relative to DMSP in experimental incubations containing filtered seawater, which we hypothesize is related to the bacterial preference for the uptake of 34S‐depleted DMS. Overall, the results indicate that plankton symbiosis can, at times, represent a potentially important source of DMS(P). Specific differences in 34S provided new insights into sulfur isotopic fractionation associated with DMS(P) biotransformation processes, with potential implications for current interpretations of isotopically tracked biogenic sources of marine aerosols.