BACKGROUND: Healthcare assistants are an important group of workers who can influence public opinion. Their attitudes toward organ donation may influence public awareness of healthcare matters; negative attitudes toward donation and transplantation could have a negative impact on public attitudes. Our objective was analyze the attitudes of healthcare assistants, in Spanish and Mexican healthcare centers toward organ donation and determine factors affecting them using a multivariate analysis. MATERIAL AND METHODS: As part of the "International Collaborative Donor Project," 32 primary care centers and 4 hospitals were selected in Spain and 5 hospitals in Mexico. A randomized sample of healthcare assistants was stratified according to healthcare services. Attitudes were evaluated using a validated questionnaire of the psychosocial aspects of donation, which was self-completed anonymously by the respondent. Statistical analysis used the chi-square test, Student t test, and logistic regression analysis. RESULTS: Of 532 respondents, 66% in favored donation and 34% were against it or undecided. Upon multivariate analysis, the following variables had the most weight: 1) country of origin (Mexicans were more in favor than Spanish; odds ratio [OR]) = 1.964; P = .014); 2) a partner with a favorable attitude (OR = 2.597; P = .013); 3) not being concerned about possible bodily mutilation after donation (OR = 2.631; P = .006); 4) preference for options apart from burial for handling the body after death (OR = 4.694; P < .001) and 5) accepting an autopsy if one was needed (OR = 3.584; P < .001). CONCLUSIONS: The attitudes of healthcare assistants toward organ donation varied considerably according to the respondent's country of origin. The psycho-social profile of a person with a positive attitude to donation was similar to that described within the general public.