Within this study, we investigated whether the polyunsaturated fatty acids (PUFA)-rich nature of rumen protozoa is a consequence of ingestion of PUFA-rich chloroplasts. Four Hereford × Friesian steers were offered hay [low 18:3 (n-3) and low chlorophyll concentration] followed by freshly cut perennial ryegrass [high 18:3 (n-3) and high chlorophyll concentration] for 16 days. On the 14th and 16th days, rumen protozoa as well as attached and planktonic bacteria were fractionated 1 h before (−1 h), 2 and 6 h postfeeding, and their fatty acid concentrations determined. Protozoa fractionated from fresh grass-fed steers were richer (P<0.05) in PUFA, except conjugated linoleic acid, for all time points compared with those from hay-fed steers. Protozoal density was higher (P<0.05) for grass compared with hay. Entodinomorphid abundance was 3.4 times higher on fresh grass (P<0.01) compared with hay. Confocal microscopy and transmission electron microscopy confirmed that Epidinium spp. were commonly saturated with intracellular cytoplasmic chloroplasts. These data suggest that engulfment of chloroplasts is a major contributor to the high 18:3 (n-3) concentration of protozoa.