The World Health Organization said Muhammad Ishaq was killed on Friday evening. In a statement Saturday, it did not specify any suspected motive.
Immunization activities in Pakistan's biggest city were suspended this week after the previous incident.
Pakistan -- one of only three countries where the disease is endemic -- is in the middle of a campaign to vaccinate children under five. Taliban militants in northern Pakistan have barred the vaccination campaign from territory under their control, saying it can't go forward until the U.S. stops drone strikes in the country.
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