Alfin1 cDNA, obtained by differential screening of a poly(A)+ library from salt-tolerant alfalfa cells, encodes a novel protein with a Cys4 and His/Cys3 putative zinc-binding domain that suggests a possible role for this protein in transcriptional regulation. We have expressed the cDNA in Escherichia coli and show that the recombinant Alfin1 protein binds DNA in a sequence-specific manner. The DNA recognition sequence was determined from individual clones isolated after four rounds of random oligonucleotide selection in gel retardation assays, coupled with PCR amplification of the selected sequences. The consensus binding site for Alfin1 is shown to contain two to five G-rich triplets with the conserved core of GNGGTG or GTGGNG in clones showing high-efficiency binding. DNA binding of the recombinant Alfin1 was inhibited by EDTA. Alfin1 mRNA was found predominantly in alfalfa roots. Growth of salt-sensitive Medicago sativa L on 171 mM NaCl led to a slight decrease in Alfin1 mRNA, while the salt-tolerant plants showed no decrease in Alfin1 mRNA levels. Interestingly, recombinant Alfin1 binds efficiently to three fragments of the MsPRP2 promoter, each containing consensus sequences identified by the random oligonucleotide selection. Since MsPRP2 transcripts were shown to be root-specific and accumulated in alfalfa roots in a salt-inducible manner, Alfin1 may play a role in the regulated expression of MsPRP2 in alfalfa roots and contribute to salt tolerance in these plants.