One of the most highly conserved steps in left–right patterning is asymmetric gene expression in lateral plate mesoderm (LPM). Here, we quantitatively describe the timing of the posterior to anterior (PA) wave-like propagation of zebrafish southpaw (Nodal) and pitx2 in LPM and lefty1 in the midline. By altering the timing of the PA wave, we provide evidence that the PA wave in the LPM instructs brain asymmetry. We find that initiation of pitx2 in LPM and lefty1 in midline depends on Southpaw, and that casanova (sox32) and two Nodal inhibitors, lefty1 and charon, have distinct roles upstream of PA wave initiation. Surprisingly, Casanova, endoderm and Kupffer's Vesicle are not required for normal timing of southpaw initiation and PA propagation. In contrast, lefty1 morphants display precocious asymmetric initiation of southpaw with an intrinsic left-hand orientation, whereas charon morphants have premature initiation without LR orientation, indicating distinct roles for these Nodal antagonists. Developmental Dynamics 237:3640–3647, 2008. © 2008 Wiley-Liss, Inc.