V-src-dependent and -independent alterations in the steady-state content, rates of synthesis, and turnover of fibronectin protein and mRNA were identified using rat fibroblasts which are temperature sensitive for p60v-src activity. Activation of p60v-src caused a fivefold reduction in the rate of fibronectin biosynthesis. The v-src-dependent decrease in fibronectin biosynthesis resulted from a similar reduction in the steady-state content of fibronectin mRNA. This change was reversible and required more than 24 h, implying an indirect effect of p60v-src on fibronectin gene expression. The rate of fibronectin mRNA turnover and pattern of alternative splicing were unchanged following p60v-src activation, indicating that these regulatory steps are insensitive to v-src transformation. A v-src-specific reduction of at least threefold was measured for the rate of fibronectin gene transcription, and gene transfer studies using fibronectin promoter-CAT reporter genes indicated that transcriptional repression occurs at the level of initiation. When p60v-src was inactive, CAT reporter genes controlled by 4.9 or 3.2 kb of the rat fibronectin promoter exhibited relatively increased CAT activity (approximately twofold) compared to another CAT reporter construction controlled by only 880 bp of the fibronectin promoter. In contrast, CAT activity was relatively reduced (approximately twofold) for the reporter constructions containing 4.9 or 3.2 kb of the promoter when p60v-src was active. These findings indicate that the distal portion of the fibronectin promoter contains a v-src-sensitive element(s) which mediates a decrease in the rate of fibronectin transcription initiation by negative control.