The in vitro activities of the 14-membered macrolides (14M) clarithromycin (CL), roxithromycin (RO), dirithromycin (DI) and flurithromycin (FL), a 15-membered macrolide (15M) azithromycin (AZ), and the 16-membered macrolides (16M) josamycin (JM), spiramycin (SP), miocamycin (MI) and rokitamycin (RK) were compared with erythromycin (ER). CL was two- to four-fold more potent than ER against all organisms except Haemophilus and Campylobacter jejuni against which it was two-fold less active. RO and DI were generally two- to four-fold less active than ER. FL was two-fold less active than ER. AZ was equal to or two-fold less active than ER against Gram-positive bacteria and two- to four-fold more active than ER against Gram-negative bacteria, such as Haemophilus, Neisseria gonorrhoeae and Branhamella. The 16M were generally four- to eight-fold less active than ER against most aerobic bacteria and two-fold less active than ER against anaerobic bacteria. RK was generally two-fold more active than the other 16M. Bacteria carrying a constitutive-type of MLS resistance were resistant to the 14M, 15M and 16M, whereas bacteria with an inducible-type of resistance were susceptible to the 16M but resistant to the 14M and 15M. Addition of serum to the medium increased the activity of ER, CL, DI, FL, AZ and SP. The activity of RO was reduced four- to six-fold and the activity of MI was reduced by two-fold by adding serum to the medium. The activity of JO was unaffected by serum. All the macrolides were one- to four-fold more active at pH 8.0 than at pH 6.5. All the macrolides were bacteriostatic against Staphylococcus aureus 553, except RO and MI which were slowly bactericidal. All the macrolides were slowly bactericidal against Haemophilus influenzae 1435.