Fibronectins (FNs) are essential for the proper development of embryonic mesenchymal tissues. A lacZ reporter gene has been fused to 4.9 kbp of DNA from the rat FN gene 58 flanking region, and this construct has been microinjected into fertilized mouse embryos to investigate the cis elements needed for the temporal and spatial regulation of FN in vivo. Histochemical staining of embryos for b-galactosidase activity demonstrated that four independent lines shared a specific pattern of lacZ expression, reflecting the activity of the fibronectin sequences contained within the transgene. Specifically, somites stained positively for lacZ, but expression was spatially and temporally non-uniform, with higher levels in more caudal somites after a total of ca. 13 somite pairs had formed. This rostral-caudal gradient of lacZ expression in somites of embryos beyond this stage resembled the distribution of endogenous FN mRNA, as detected by whole mount in situ hybridization. The transgene was not expressed in the developing heart where endogenous FN mRNA was detected. Unexpectedly, highly localized staining was observed within the neural tube beginning at ca. E10-10.5, and two of the lines exhibited additional areas of staining due to the individual integration sites. Thus, the 4.9 kbp FN fragment appears to recapitulate closely the complex pattern of FN expression observed during somitogenesis. A smaller fragment of 0.9 kbp also directed lacZ expression in caudal somites at E9.5, suggesting that these sequences are sufficient to establish the spatio-temporal pattern.