Polyunsaturated fatty acids (PUFAs) are abundant in stream-dwelling aquatic insects and might be essential for their growth and survival. We conducted a controlled study designed to clarify fatty acid (FA) assimilation and metabolism by net-spinning caddisfly larvae (Hydropsyche sp.). Specifically, our goal was to determine if these insects could synthesize essential FAs (18:2ω6, 18:3ω3, 20:4ω6, and 20:5ω3) de novo or elongate them from precursors, or if they must acquire them from their diet. In laboratory experiments, we supplied controlled diets (commercial fish food, oat particles, Cladophora glomerata, and conditioned leaves) to larvae in individual microcosms that simulated stream conditions. Caddisfly FA profiles consistently matched those of the diets provided, and in general, these insects had very limited abilities to synthesize and elongate essential FAs. Concentrations of linoleic (18:2ω6) and linolenic (18:3ω3) acid declined significantly in caddisflies fed diets depleted in these FAs. To test the ability to elongate FAs, caddisflies were fed a diet depleted in arachidonic acid (20:4ω6), but rich in its precursor 18:2ω6. Tissue concentrations of 20:4ω6 declined, suggesting that the ability of caddis larvae to elongate 18:2ω6 to 20:4ω6 is limited or absent. Caddisflies accumulated 20:5ω3 at greater assimilation efficiencies than for any other FA, suggesting a key importance of this FA. Across all experiments, caddisflies gained mass, total FA content, or both when supplied with all food sources except leaf litter. We suggest that caddisflies in streams must obtain essential FAs either from algal material or from predation.