In mammalian cerebrum there exist two distinct types of interneurons expressing nitric oxide synthase (NOS). Type I neurons are large in size and exhibit heavy nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical reaction, while type II cells are small with light NADPH-d reactivity. The time of origin of these cortical neurons relative to corticogenesis remains largely unclear among mammals. Here we explored this issue in guinea pigs using cell birth dating and double labeling methods. Bromodeoxyuridine (BrdU) pulse-chasing (2 doses at 50 mg/kg, 12 h apart) was given to time-pregnant mothers, followed by quantification of NADPH-d/BrdU colocalization in the parietal and temporal neocortex in offspring at postnatal day 0 (PO), P30 and P60. Type I neurons were partially colabeled with BrdU at PO, P30 and P60 following pulsechasing at embryonic day 21 (E21), E28 and E35, varied from 2-11.3% of total population of these neurons for the three time groups. Type II neurons were partially colabeled for BrdU following pulse-chasing at E21, E28, E35 and E42 at PO (8.6%-16.5% of total population for individual time groups). At P60, type II neurons were found to co-express BrdU (4.8-11.3% of total population for individual time groups) following pulse chasing at E21, E28, E35, E42, E49, E56 and E60/61. These results indicate that in guinea pigs type I neurons are generated during early corticogenesis, whereas type II cells are produced over a wide prenatal time window persisting until birth. The data also suggest that type II nitrinergic neurons may undergo a period of development/differentiation, for over 1 month, before being NADPH-d reactive.