We determined the protein composition and antigenic content of saliva from Amblyomma hebraeum female ticks of different weight classes. The mean protein concentration of saliva of small partially fed ticks (< 100 mg) was 333 +/- 83 micrograms/ml and that of large partially fed ticks (150-420 mg) was 59 +/- 14 micrograms/ml. The reduction in concentration mostly was caused by the significantly higher fluid volume per minute secreted by large ticks. Polypeptide analysis of saliva indicated the presence of a protein (14 kilodaltons [kD]) only in ticks weighing < 60 mg. Other saliva proteins of 21 and 26 kD were present only in ticks weighing < 150 mg, whereas 68-kD protein was absent or very faint in ticks > 100 mg. Immunoblot analysis indicated that sera from rabbits infested with ticks recognized 13 saliva antigens ranging in size from 23 to 200 kD. The antigens were present in detectable quantities in the saliva of small ticks only. In contrast, the sera from rabbits immunized with tick saliva recognized only four antigens from 63 to 200 kD. The 63-kD antigen was not present in the saliva of large ticks. These data indicate that the saliva of A. hebraeum from small ticks is antigenically more complex than that of large ticks and that the route of immunization influences the humoral immune response of the host to the saliva antigens.