Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.