GDP-D-arabinopyranose (GDP-D-Ara) is the precursor of the uncommon D-arabinopyranose residues present in the glycoconjugates of a few trypanosomatid parasites. Biosynthetic labelling experiments with Crithidia fasciculata showed that GDP-D-Ara could be labelled with [3H]D-Ara, [2-3H]D-Glc and [6-3H]D-Glc, but not with [1-3H]D-Glc, suggesting that D-Ara can be either taken up directly by the parasite or derived from D-Glc through a pathway involving the loss of carbon C-1. In vivo pulse-chase experiments indicated that D-Ara was sequentially incorporated into D-Ara-1-PO4 and GDP-D-Ara prior to transfer to the acceptor glycoconjugate, lipoarabinogalactan. An MgATP-dependent D-arabino-1-kinase activity present in soluble extracts of C. fasciculata was purified away from phosphatase activities by size-exclusion chromatography. The D-arabino-1-kinase had an apparent molecular mass of 600 kDa, a neutral optimum pH, and displayed substrate inhibition at D-Ara concentrations above 100 microM. It had a KmATP of 1.7 mM and a KmAra of 24 microM. Competition studies indicated that the orientation of every single hydroxyl residue was important for D-Ara recognition by the enzyme, but that methyl or hydroxymethyl groups could be tolerated as equatorial substituents on C-5 of D-Ara. The partially purified D-arabino-1-kinase activity was used in the chemico-enzymic synthesis of GDP-[5-3H]D-Ara from [6-3H]D-GlcN.