The design, synthesis, and receptor subtype binding affinities of several 2-substituted thioadenosine nucleoside and nucleotide analogues are described. Alkylation of 2-thioadenosine (1) with iodopentenylboronic acid followed by iododeboronation gave 2-((E)-1-iodo-1-penten-5-yl) thioadenosine (9). Compound 1 on treatment with 4-nitrobenzyl bromide and propargyl bromide furnished compounds 3 and 5, respectively. The 5'-monophosphate analogues of compounds 3, 5, 7, and 9 were prepared similarly using 2-thioadenosine 5'-monophosphate (2). Treatment of 1 with bromoethylamine hydrobromide provided 2-[(aminoethyl)thio]adenosine (11) which on coupling with N-succinimidyl 3-(4-hydroxyphenyl)propionate gave 2-[[[3-(4-hydroxyphenyl)propionamido]ethyl]thio]adenosine (12). Iodination of 12 gave 2-[[[3-(4-hydroxy-3-iodophenyl)propionamido]ethyl]thio]adenosine (13). Compounds 3-13 were evaluated for their affinities toward A1 and A2 adenosine receptors in rat brain cortex and striatum, respectively using [3H]DPCPX and [3H]CGS21680 as ligands. The nucleotide analogues 4, 6, 8, and 10 inhibited binding of [3H]DPCPX by 10-20% and of [3H]CGS21680 by 40-50% at a concentration of 100 microM suggesting weak affinity toward adenosine receptors. The nucleoside analogues 3, 5, 7, 9, 12, and 13 inhibited the A2 receptor binding of [3H]CGS21680 with Ki values of 1.2-3.67 microM, while A1 receptor binding of [3H]DPCPX was inhibited with Ki values 10-17 microM. The A1/A2 ratios suggest 4-8-fold A2 receptor selectivity.