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— Although the effects of ultraviolet B (UVB, 290–320 nm) radiation have been studied in plants extensively, little is known about the potential impacts on maturation of chloroplasts. To address this problem, the effects of supplementary UVB on chloroplast development were examined in the aquatic higher plant Spirodela oligorrhiza. Dark-grown Spirodela-containing proplastids were exposed to photosynthetically active radiation (PAR) and ultraviolet A (UVA, 320–400 nm), plus supplementary UVB equivalent to 1% of PAR on a photon basis. The biosynthesis and assembly of chlorophyll (Chi) into reaction centers was followed for 4 days in situ by low temperature (77 K) Chi fluorescence. Impacts on chloroplast development were detected after only 1 h incubation in light with supplementary UVB. Fluorescence emission signals from Chi associated with the photosystem (PS) II antenna, PSII reaction centers and PSI reaction centers were detected at the same time with or without UVB, but the magnitude of PS fluorescence was diminished up to 60% in plants incubated in UVB. The Chi content was also lower in UVB-treated plants, but to a lesser degree than anticipated by low temperature fluorescence, suggesting lack of organization and/or association of Chi with PS. Electron transport, measured with room temperature fluorescence induction, was not consistently different in plants exposed to UVB. These results suggest that with UVB, fewer and/or smaller PS form during chloroplast development, but there is not a large inhibition of Chi synthesis or PSII activity.