Abstract. Periphytic algae attached to a streambed substrate (periphyton) are an important primary producer in stream ecosystems. We determined the isotopic composition of chlorophyll a in periphyton collected from a stream flowing on limestone bedrock in the Seri River, central Japan. Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) and natural radiocarbon abundances (Δ¹⁴C) were measured in chlorophyll a (δ¹³C_chl, δ¹⁵N_chl and Δ¹⁴C_chl) and bulk (δ¹³C_bulk, δ¹⁵N_bulk and Δ¹⁴C_bulk) for periphyton, a pure aquatic primary producer (Cladophora sp.) and a terrestrial primary producer (Quercus glauca). Periphyton δ¹³C_bulk and δ¹³C_chl values did not necessarily correspond to δ¹³C_bulk for an algal-grazing specialist (Epeorus latifolium). Periphyton Δ¹⁴C_chl values (−258 ‰ in April and −190 ‰ in October) were slightly lower than Δ¹⁴C_bulk values (−228 ‰ in April and −179 ‰ in October) but were close to the Δ¹⁴C value for dissolved inorganic carbon (DIC; −217 ± 31 ‰), which is a mixture of weathered carbonates (Δ¹⁴C = −1000 ‰), CO₂ derived from aquatic and terrestrial organic matters (variable Δ¹⁴C) and dissolved atmospheric CO₂ (Δ¹⁴C approximately +30 ‰ in 2013). Δ¹⁴C_chl values were also close to Δ¹⁴C_bulk for E. latifolium (−215 ‰ in April and −199 ‰ in October) and Cladophora sp. (−210 ‰), whereas the Δ¹⁴C_bulk value for Q. glauca (+27 ‰) was closer to Δ¹⁴C for atmospheric CO₂. Although the bulk isotopic composition of periphyton is recognised as a surrogate for the photosynthetic algal community, natural periphyton is a mixture of aquatic and terrestrial organic materials. Our results indicate that the bulk periphyton matrix at the study site consists of 89 to 95 % algal carbon (derived from ¹⁴C-depleted DIC) and 5 to 11 % terrestrial organic carbon (derived from ¹⁴C-enriched atmospheric CO₂).