Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium ('flat'). When anthers were plated with only one loculus in contact with the medium ('up'), a high proportion of the anthers produced calluses. The top loculus of the 'up' anthers was most productive. 'Flat' anthers, when compared with 'up' anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in 'flat' anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.