The interaction of antibodies directed toward N6-(delta2-isopentenyl)adenosine, i6Ado, or its nucleotide with related bases, nucleosides, nucleotides and purified tRNAs is described. The selectivity of the antibody preparation was tested in inhibition experiments utilizing a sensitive radioimmunoassay to quantitate the binding of [3H]i6Ado to the antibody. Purified tRNAs containing various modified nucleosides adjacent to the 3'-end of the anticodon were tested to provide information about the selectivity of the antibody preparation toward nucleotides in this position of the tRNA chain. Antibodies directed against the nucleotide hapten were used to purify tRNAs which contain i6Ado and to quantitate the amount of that nucleotide. The same order of selectivity was expressed whether the nucleotides were free or in a tRNA molecule. Interaction of the antibody with compounds from the i6Ado family demonstrated dominance of the hydrophobic isopentenyl group and the importance of positional differences of modifications.