None of those infected - mentally ill patients and prisoners - consented.
Guatemalan President Alvaro Colom accused the US of "crimes against humanity".
US President Barack Obama has called Mr Colom to apologise and has said the acts ran contrary to American values.
“
Start Quote
We deeply regret that it happened, and we apologise to all the individuals who were affected by such abhorrent research practices”
End Quote
Statement from secretaries of state and health
'Shocking, tragic, reprehensible'
Syphilis can cause heart problems, blindness, mental illness and even death, and although the patients were treated it is not known how many recovered.
Evidence of the programme was unearthed by Prof Susan Reverby at Wellesley College. She says the Guatemalan government gave permission for the tests.
No offer of compensation has yet been made, but an investigation will be launched into the specifics of the study, which took place between 1946 and 1948.
White House spokesman Robert Gibbs said on Friday the news was "shocking, it's tragic, it's reprehensible".
The joint statement from Mrs Clinton and Health Secretary Kathleen Sebelius said: "Although these events occurred more than 64 years ago, we are outraged that such reprehensible research could have occurred under the guise of public health.
"We deeply regret that it happened, and we apologise to all the individuals who were affected by such abhorrent research practices."
Unaware
The study by Prof Reverby shows that US government medical researchers infected almost 700 people in Guatemala with two segxwally transmitted diseases.
The patients - prisoners and people suffering mental health problems - were unaware they were being experimented upon.
The doctors used prostitutes with syphilis to infect them, or inoculation, as they tried to determine whether penicillin could prevent syphilis, not just cure it.
The patients were then treated for the disease, but it is unclear whether everyone was cured.
Prof Reverby has previously done research on the Tuskegee experiment, where the US authorities measured the progress of syphilis in African-American men without telling them they had the disease or adequately treating it.
The experiment ran from 1932 to 1972, with President Bill Clinton eventually apologising for it.
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