Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence analysis of SCO5208 and SCO2771, amplified from the HolPase mutant strain, revealed that SCO5208 shows a mutation in a conserved amino acid, whereas SCO2771 does not show any mutation. All these results show that S. coelicolor SCO5208, recently renamed hisN, is the HolPase involved in histidine biosynthesis.