Afferent projections from the periaqueductal gray matter (PAG) to the B3 region (nucleus raphe magnus, NRM; and nucleus reticularis paragigantocellularis, NPG) were examined by means of the horseradish peroxidase (HRP) method revealed by using the tetramethylbenzidine (TMB) procedure. Following iontophoretic injection of HRP into the B3 cellular area, numerous labeled neurons were found between the third oculomotor complex and the rostral part of the tegmental nucleus dorsalis of Gudden. The most densely labeled regions were the lateral parts of the dorsal raphe nucleus (wings of the DRN) and Hamilton's nucleus dorsalis of the PAG. Very few neurons were found within the medial part of the DRN. In contrast, results with control injections performed outside the B3 area gave a different distribution of labeled neurons. The functional significance of these connections, particularly those emanating from the wings of the DRN which represent new information, is briefly discussed with regard to mechanisms operative in the control of pain. Although the major PAG-B3 direct projections could underlie a role for the B3 area in PAG-induced analgesia, the comparison of our anatomical and behavioral results points out some problems. Indeed, there is no strict correlation between the location of stimulation sites which induce analgesia in the freely moving rats and the distribution within the PAG of B3 projections. These observations lead us to question the role of direct PAG-B3 connections in the antinociception induced by PAG stimulation and alternative hypotheses are proposed.