Introduction: Spermatozoa discard excessive cytoplasm during spermiogenesis. In the elongated spermatids, most of cytoplasm is isolated in the midpiece with the posterior ring and the annulus. Prior to spermiation, excessive cytoplasm migrates to the neck, and is pinched off as the residual body. The remnant of cytoplasm left at the neck is called the cytoplasmic droplet. During epididymal passage of spermatozoa, the droplet migrates distally toward the annulus along the mitochondrial sheath. This is a common phenomenon in most mammalian species, but the migration mechanism of the droplet has not been solved. To get a clue, we examined guinea pig spermatozoa, because they show following characteristics: (1) the droplet stops at the central portion of the midpiece in matured spermatozoa, and (2) by a freeze replica method, linear arrays of intramembranous particles are detected on the plasma membrane at the site where it closely apposed to the outer mitochondrial membrane. This suggests that the arrays show the adhesion site of the two membranes in two dimensional view. Materials and Methods: The epididymides of adult male guinea pigs were fixed with 2.5 % glutaraldehyde and were separated into 6 segments. The luminal spermatozoa of each segment were examined by thin sectioning electron microscopy after tannic acid treatment, and by freeze-replicas method. Results: All droplets situated at the neck until spermatozoa came down to segment 3. In segments 4-6, the droplets migrated to the center of the midpiece in 3/4 spermatozoa, while they were left at the neck in the rest of them. In segments 1-3 spermatozoa, the space between the plasma and outer mitochondrial membranes varied from 20nm to 70nm at the midpiece distal to the droplet. While in segments 4-6 spermatozoa, the space became narrower and regular, and was 15.85nm (SD=1.74nm: n=10) throughout the midpiece, except at the droplet, where the two membranes were widely separated. The narrow space was filled with dark substance, and negatively stained thin fibrils connected the two membranes. In freeze replica images, the linear arrays on the plasma membrane first appeared in segments 2-3 spermatozoa. The arrays were variously developed in the distal midpiece, while in the proximal midpiece they were limited in narrow area adjacent to the posterior ring. Segments 4-6 spermatozoa had well developed array both in proximal and distal area to the droplet. While the arrays poorly developed on the plasma membrane covering the droplet irrespective to its position. Discussion: Present observations revealed that formation of the linear arrays was a first sign of tight adhesion between the plasma and outer mitochondrial membranes. Adhesion from the proximal side may push the droplet distally, while distal adhesion prevents further migration of the droplet. In guinea pig spermatozoa, balance between pushing and stopping force determines the final position of the droplet in the midpiece. Regular double-helix arrangement of the mitochondria must be significant for up and down movement of residual cytoplasm and for the tight adhesion of the two membranes. Failure of this membrane adhesion may cause some tail abnormalities, such as coiled tail and hairpin tail. Conclusion: Adhesion between the plasma and outer mitoshondrial membranes is involved in the cytoplasmic droplet migration at least in guinea pig spermatozoa.