The Agrobacterium-mediated transformation system was extended to a famous Javanica rice variety, Rojolele, that is cultivated in Indonesia now. Efficient callus induction from immature and mature seeds of Rojolele did not succeed by any previous method for any rice cultivar. In this study, the callus from mature seeds of Rojolele exhibited a compact and nodular appearance on C medium after the carbon source and medium pH was modified. Scutellum-derived calli from mature seeds were co-cultivated with Agrobacterium tumefaciens strains EHA101 or LBA4404 that carried plasmid pAFT14, which contained the genes for beta-glucuronidase (gus) and hygromycin resistance (hpt). Finally, the transformation efficiency of Rojolele variety using A. tumefaciens strain EHA101 (pAFT14) was improved to about 23%, similar to that of the Japonica rice variety Nipponbare. The seed fertility of transgenic Rojolele was more than 90%. The copy number of the transgene varied from one to three copies in the T(0) transgenic lines. Both the gus and the hpt genes were inherited and expressed in the progeny.