Gelatin-coated slides were used to obtain en face preparations of retinal pigment epithelium (RPE) from 6- to 21-day-old chick embryos in order to study the distribution of F-actin in microfilaments (MF) and the MF-associated proteins, myosin, tropomyosin, alpha-actinin and vinculin in situ at different stages of development by fluorescence microscopy. The epithelial sheets were fixed in formaldehyde and then extracted in a solution containing 0.1% Triton X-100. NBD-Phallacidin was used to visualize the F-actin in MF, and antisera against myosin, tropomyosin, alpha-actinin and vinculin were used to determine the distribution of these four MF-associated proteins. F-actin, myosin, tropomyosin, alpha-actinin and vinculin were present in cortical rings around the apical ends of the RPE cells throughout this period of development. Of these proteins, only F-actin was identified in the apical processes of RPE cells. The increase in the amount of F-actin could be followed as the length and the number of apical processes increased with age and maturation of RPE cells. F-actin was first detected in numerous short apical processes on the surface of each RPE cell on day 12. From day 12 to day 17, they were at an intermediate stage of elongation and from day 17 onward all of the RPE cells had long F-actin-containing apical processes. These results indicate that the F-actin-containing MF assemble much later in the apical processes than in the cortical rings.(ABSTRACT TRUNCATED AT 250 WORDS)