The relationship of current use of cigarettes, marijuana and alcohol to the parameters of seminal fluid analysis, sperm penetration assay and sperm autoimmunity was studied in 164 men from infertile couples. Current cigarette smokers, marijuana users and heavy alcohol users showed greater numbers of leukocytes in the seminal fluid than did nonusers (p less than 0.02, less than 0.007 and less than 0.01, respectively). In addition, cigarette smokers had lower sperm penetration assay scores than nonsmokers (median 2.5 versus 8.0, p = 0.05). Users of cigarettes, marijuana or alcohol showed no decrease in sperm count, motility or percentage of oval sperm, and no difference in prevalence of antisperm antibodies compared to nonusers. After controlling for past sexually transmitted diseases and multiple substance exposures in a multivariate model, use of cigarettes (p = 0.006), marijuana (p = 0.12) or alcohol (p = 0.098) continued to be associated with a trend toward increased number of seminal fluid leukocytes. Cigarette smoking continued to show a significant decrease in sperm penetration assay score (p = 0.03).