In the present study, cis elements in the 5'-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating basal and alpha 1-adrenergic-inducible transcription were investigated. Truncation analyses using ANF-luciferase reporter constructs transfected into primary neonatal rat cardiac myocytes showed that an A/T-rich serum response element (SRE) at -114 bp of the ANF 5'-FS, which bound serum response factor (SRF), was required for basal and inducible transcription. In constructs composed of 134 bp of rat ANF 5'-FS driving luciferase (ANF-134Luc), mutations in the SRE at -114 bp disrupted SRF binding and ANF promoter activity. However, the same mutations in ANF-638Luc had little effect, suggesting a collaborating role for more distal sequences, such as the other SRE in ANF-638 at -406 bp. In ANF-638Luc, mutations in the SRE at -406 bp that disrupted SRF binding to that site decreased ANF reporter activity by only 25%; however, mutating both of the SREs completely blocked alpha 1-adrenergic-inducible activity. Mutation analyses showed that an … (SP-1)-like site at -69 bp, shown previously to confer inducibility in reporters with 134 bp of ANF 5'-FS, was not required in ANF-638Luc. However, double mutants in the SP-1-like region and either SRE completely blocked alpha 1-adrenergic-inducible ANF promoter activity. These findings emphasize that no single element is responsible for alpha 1-adrenergic agonist-regulated ANF transcription but that the SREs at -114 and -406 bp and the SP-1-like sequence at -69 bp mediate the effect in collaboration.