RNA-binding proteins such as heterogeneous nuclear ribonucleoproteins (hnRNPs), which contain the bulk of methylated arginine residues in eukaryotic cells, play many essential roles in the metabolism of nuclear pre-mRNA. Arginine methyltransferase activity has also been implicated in signal transduction events with components of the cellular growth and viral response pathways. We recently characterized a single yeast hnRNP methyltransferase (HMT1). We now present the identification and characterization of two putative human arginine methyltransferases termed HRMT1L1 and HRMT1L2. In addition to methyltransferase similarities, the N-terminal region of the HRMT1L1 protein contains an Src homology 2 domain. HRMT1L1 maps to a YAC containing the telomere of chromosome 21q. Three alternatively spliced HRMT1L2 transcripts with variable 5'-ends were observed, encoding proteins of 343, 347, and 361 amino acids, respectively. HRMT1L2 maps to human chromosome 19q. Recombinant HRMT1L2 protein encoded by the most common 5'-variant exhibited methyltransferase activity in vitro. Furthermore, in vivo activity was demonstrated by complementation of a yeast HMT1 mutant strain. The identification of highly conserved Hmt1p human homologues that function in yeast indicates that analyses of this class of enzymes in yeast may be directly applicable to higher eukaryotes. The possible roles of HRMT1L1 and HRMT1L2 in human disease are currently unknown.