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 α₁-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 α₁-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 α₁-adrenergic–inducible ANF promoter activity. These findings emphasize that no single element is responsible for α₁-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.