The cyl genes of Streptococcus agalactiae are required for the production of hemolysin. Based on the observation that nonhemolytic S. agalactiae mutants do not produce pigment, a close genetic linkage between hemolysin and pigment has been postulated. To investigate this genetic linkage and to identify genes involved in the production of the S. agalactiae pigment, we screened mutant libraries for nonpigmented clones. Four distinct mutants were isolated with a nonpigmented and nonhemolytic phenotype. The mutations had occurred either in known cyl genes or in two open reading frames located immediately downstream. These novel genes are cotranscribed with the cyl gene cluster and were designated cylF and cylI. Our data indicate that identical genes participate in the production of S. agalactiae hemolysin and pigment.