Nitric oxide (NO) enhances prolactin-stimulated DNA synthesis and inhibits prolactin-induced differentiation in mouse mammary epithelium. The molecular pathways used by NO were determined by employing specific inhibitors of the transducers utilized by NO. Inhibitors of the Jun N-terminal kinase (JNK) blocked the effect of NO on DNA synthesis, although this appeared to involve a protein kinase G (PKG)-independent pathway. In contrast, inhibitors of the extracellular signal-regulated kinase (ERK) prevented NO from suppressing alpha-lactalbumin accumulation and this effect was PKG-dependent. NO can also elevate cAMP through the inhibition of phosphodiesterase 3 and cAMP mimicks the actions of NO on both DNA synthesis and differentiation. However, suppression of cAMP levels did not prevent the effects of NO. Therefore, NO uses two separate pathways to affect mammary epithelium: it stimulates growth via JNK and inhibits differentiation through ERK. (C) 2003 Elsevier Science (USA). All rights reserved.