The heterogeneous nuclear ribonucleoprotein (hnRNP) family includes predominantly nuclear proteins acting at different stages of mRNA metabolism. A characteristic feature of hnRNPs is to undergo post-translational asymmetric arginine methylation catalysed by different type 1 protein arginine methyltransferases (PRMTs). A novel mammalian hnRNP, E1B-AP5, recently identified by its interaction with adenovirus early protein E1B-55 kDa, has been proposed to have a regulatory role in adenoviral and host-cell mRNA processing/nuclear export [Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J. Virol. 72, 7960-7971]. Here we report that E1B-AP5 is methylated in vivo in its Arg-Gly-Gly (RGG)-box domain, known to mediate protein-RNA interactions. The activity responsible for E1B-AP5 methylation forms a complex with E1B-AP5 in vivo. The predominant mammalian arginine methyltransferase HRMT1L2 (hPRMT1) did not detectably methylate endogenous E1B-AP5 despite efficiently methylating a recombinant RGG-box domain of E1B-AP5. Using yeast two-hybrid screening we identified HRMT1L1 (PRMT2) as one of the proteins interacting with E1B-AP5. By in situ immunofluorescence we demonstrated that E1B-AP5 co-localizes with the nuclear fraction of HRMT1L1. The Src homology 3 (SH3) domain of HRMT1L1 was essential for its interaction with E1B-AP5 in vivo. We suggest that HRMT1L1 is responsible for specific E1B-AP5 methylation in vivo.