The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a 3D model of the Aspergillus nidulans purine-cytosine/H+ FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1-5 and TMS6-10), which are followed by two additional TMSs. TMS1, TMS3, TMS6 and TMS8 form an open cavity, which is predicted to host the substrate binding site. Based on primary sequence alignment, 3D topology and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; S85 (TMS1), W159, N163 (TMS3), W259 (TMS6) and N354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus N350, N351 and P353 are irreplaceable for FcyB function. Among these residues, S85, N163, N350, N351 and N354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that S85, N163 and N354 directly interact with substrates, W159 and W259 stabilise binding through pi-pi stacking interactions, P353 affects the local architecture of substrate binding site, whereas N350 and N351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family, by combining genetic and computational approaches.