Arachidonic acid (ARA) is essential in felines because conversion of dietary linoleic acid (LA) to ARA is rate-limited by low Δ6-desaturase. Dietary γ-linolenic acid (GLA) may serve as an ARA precursor by-passing this initial rate-limiting step. This possibility was investigated using twenty-six adult female domestic shorthair cats divided into three groups and fed on complete and balanced diets containing high GLA (GL), high LA (HL) or low LA (LL, control) diets, for 300 d prior to ovariohysterectomy. Plasma was obtained 1–2 d before surgery and uterine, ovarian and associated adipose tissues were reserved for lipid analysis. Fatty acid profiles of the plasma phospholipid (PL) fractions and adipose lipids were performed. In the GL group, plasma and uterine tissue PL were significantly enriched in GLA, di-homo GLA (DGLA) and ARA compared with control. However, ovarian and adipose tissue PL were only enriched in DGLA. Enrichment of uterine tissues with DGLA and ARA probably supplies the essential eicosanoid precursors for reproduction when GLA is fed consistently with an active Δ5-desaturase in uterus. By contrast, this enzyme appears less active in ovary because ARA was not higher compared with control. Earlier reports concluded that ARA was not necessary for fertilisation (an ovarian function), but required for successful pregnancy and reproduction (a uterine function). Adipose tissue DGLA may be a reservoir for ARA synthesis by other tissues upon mobilisation. Dietary GLA may meet feline ARA requirements in the absence of an animal-based preformed source of ARA.