Φ1207.3 is a prophage of Streptococcus pyogenes which carries the macrolide efflux resistance genes mef(A)/msr(D). Complete nucleotide sequence showed that Φ1207.3 is 52,491 bp in length and contained 58 ORFs. Φ1207.3 codes for two different C-methylation systems, several phage structural genes, a lysis cassette, and three site-specific resolvases of the serine recombinase family. The aim of this study was to characterize Φ1207.3 in a Streptococcus pneumoniae model strain obtained by mating the clinical S. pyogenes 2812A strain carrying Φ1207.3 with the laboratory S. pneumoniae FP10 strain, which is prophage-free and engineered to lack natural competence for transformation. Φ1207.3 lytic cycle was induced in the early exponential phase of pneumococcal growth with 100 ng/ml of mitomycin C for 2 hours. Concentrated culture supernatants, prepared after mitomycin C treatment, were used for Real Time-PCR analysis and for transmission electron microscope (TEM) imaging after standard negative stain. TEM showed the presence phage particles with an icosahedral head (62 nm in diameter) and a noncontractile tail (175 nm long), placing them in the Siphoviridae family. Real Time-PCR showed that mef(A) gene and circular phage genomes were enriched in the concentrated supernatants compared to the bacterial culture, while the chromosomal gyrB gene was depleted. Transduction of Φ1207.3 genome was obtained in S. pneumoniae and S. pyogenes at a frequency of 1 x 10-6 transductants/recipient cell. We could not detect plaque formation using S. pneumoniae, S. pyogenes or Streptococcus gordonii as indicator strains. In conclusion, we showed that Φ1207.3 is a phage belonging to the Siphoviridae family. The phage can be transferred from S. pyogenes to S. pneumoniae where it is still functional, albeit producing a small number of viral particles with a limited infection potential. This is the first demonstration of a functional phage carrying an antibiotic resistance determinant which is transferable among different species.