Cyclic ADP-ribose mobilizes intracellular Ca2+ in a variety of cells. To elucidate the nature of the interaction between the C3' substituent of cADP-ribose and the cADPR receptor, three analogues of NAD+ modified in the adenosine ribase (xyloNAD+ 3'F-xyloNAD+ and 3'F-NAD+ were chemically synthesised from D-xylose and adenine starting materials. 3'F-NAD+ was readily converted to cyclic 3'F-ADP ribose by the action of the cyclase enzyme derived from the mollusc Aplysia californica. XyloNAD+ and 3'F-xyloNAD+ were cyclised only reluctantly and in poor yield to afford unstable cyclic products. Biological evaluation of cyclic 3'F-ADP ribose for calcium release in sea urchin egg homogenate gave an EC(50) of 1.5+/-0.5 microM. This high value suggests that the ability of the C3' substituent to donate a hydrogen bond is crucial for agonism.