SCO3900, a putative target gene of the developmental regulator WhiI of Streptomyces coelicolor, encodes a protein similar to PadR from Pediococcus pentosaceus. The SCO3900 disruption mutant exhibited a bald phenotype with sparse aerial hyphae. However, a single copy of SCO3900 under the control of its native promoter could not complement the mutant phenotype. A fragment containing SCO3900 and its downstream three genes completely restored the mutant phenotype to the wild-type. These suggested that the disruption of SCO3900 exerted a polar effect on its downstream genes. The co-transcription of SCO3899, SCO3898, and SCO3897 with SCO3900 was confirmed by the RT-PCR. Moreover, overexpression of SCO3900 in the wild-type strain caused the same phenotype as that of the disruption mutant. The results suggested that SCO3900 encodes a negative regulator participating in morphological differentiation of S. coelicolor by influencing the expression of its downstream genes.