Acidithiobacillus ferrooxidans, an acidophilic, chemolithotrophic, γ-proteobacterium, is involved in the bioleaching of metal sulfides. For this process, bacterial attachment to mineral surface and biofilm development play a pivotal role. Generally, biofilm formation and production of exopolysaccharides is regulated by the second messenger cyclic diguanylic acid (c-di-GMP) whose cellular level depends on the synthesis and degradation activities of diguanylate cyclase (DGCs, with GGDEF domain) and phosphodiesterase (PDE, with EAL or HD-GYP domains), respectively. The analysis of the genomic sequence of A. ferrooxidans ATCC 23270 allowed us to identify 5 putative orfs encoding DGC and/or PDE-like proteins. Four of them encode for bifunctional putative proteins with GGDEF and EAL domains and are named AFE_0053, AFE_1360, AFE_1373 and AFE_1379. The fifth one named AFE_1852 has an EAL domain. The putative proteins also include PAS and GAF domains involved in signal transduction. These features suggest an involvement in signalling transduction through the metabolism of c-di-GMP. The amino acid sequences of these putative proteins were aligned with known DGCs and PDEs. Alignments indicate that AFE_1360 and AFE_1373 share more consensus sequences with active PDEs, whereas AFE_0053 and AFE_1379 do with active DGCs. On the other hand, in AFE_1852 some conserved residues of known active PDEs are changed. RT-PCR-experiments revealed that the genes that encode for these putative DGCs and/or PDEs are expressed by growth on two different substrates. These preliminary results suggest that A. ferrooxidans possesses a c-di-GMP pathway that should be involved in biofilm formation, as it occurs in many bacteria.