LIM homeodomain transcription factors play crucial roles in determining diverse aspects of neuronal development both in vertebrates and invertebrates. In the present study, we studied the expression pattern of Islet-1 (Isl-1), a member of the LIM homeodomain protein family, in the rat striatum during development. The developmental expression of Isl-1 in the striatum is highly dynamic and complex in terms of spatial and temporal regulation. The reverse transcription-polymerase chain reaction and ribonuclease protection assays demonstrated that Isl-1 messenger RNA was expressed in the developing striatum. The immunocytochemical study of Isl-1 protein expression showed that there were prominent mediolateral and caudorostral Isl-1 gradients in the developing striatum. Numerous Isl-1-positive cells appeared in the medial mantle zone of the developing striatal proper, and they co-expressed the postmitotic neuronal marker, microtubule-associated protein 2. The numbers of Isl-1-positive cells were decreased from the medial to the lateral regions, so that there were only a few Isl-1-positive cells scattered in the lateral striatum. These scattered Isl-1-positive cells were doubly labeled with tyrosine kinase receptor A and choline acetyltransferase, which indicated that they were cholinergic neurons. The Isl-1 gradients were most prominent in the embryonic day 18 and 20 striatum. With increases of time, the Isl-1 gradients were gradually reduced, and the gradients disappeared by postnatal day 7. Despite the general down-regulation of striatal Isl-1, a few Isl-1-positive cells were sustained into the adult striatum in which Isl-1 was nearly exclusively expressed by all cholinergic neurons and vice versa. Our study suggests that Isl-1 is likely to be initially expressed by postmitotic cholinergic precursors and some, if not all, non-cholinergic precursors in the developing striatum. During the progression of striatal differentiation, Isl-1 is down-regulated in non-cholinergic cells, but is sustained in cholinergic cells. The developmental restriction of Isl-1 to cholinergic neurons in the striatum may represent a novel mechanism by which LIM homeodomain proteins specify specific cell types in the striatum during development.