Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an α-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 3′-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 2′-fluoro A, but not with 2′-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C2′-endo conformation. In other cases where there is interference with 2′-deoxy A, the presence or absence of 2′-fluoro A interference helps to establish whether the 2′-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.